Polymer

ABSTRACT

The application provides a method of producing a comb polymer comprising the steps of:
         (a) Providing:
           (i) a plurality of monomers which are linear, branched or star-shaped, substituted or non-substituted, and have an olefinically unsaturated moiety, the olefinically unsaturated moiety being capable of undergoing addition polymerisation;   (ii) an initiator compound; the initiator compound comprising a homolytically cleavable bond.   (iii) a catalyst capable of catalysing the polymerisation of the monomer; and   
           (b) Causing the catalyst to catalyse, in combination with the initiator, the polymerisation of a plurality of the monomers to produce the comb polymer.       

     Catalysts and polymers obtainable by the process are also provided. 
     Preferably, the comb polymer is capable of binding proteins and may be produced from monomers which are alkoxy polyethers, such as poly(alkyleneglycol) or polytetrahydrofuran.

RELATED APPLICATION(S)

This application is a continuation of U.S. application Ser. No.11/498,525, filed Aug. 3, 2006, which is a continuation of U.S.application Ser. No. 11/311,922, filed Dec. 19, 2005 (Abandoned), whichis a continuation of International Application No. PCT/GB2004/002894,which designated the United States and was filed on Jul. 6, 2004,published in English, which is a continuation-in-part of InternationalApplication No. PCT/GB2004/002608, which designated the United Statesand was filed on Jun. 18, 2004, published in English. This applicationclaims priority under 35 U.S.C. §119 or 365 to Great Britain,Application No. GB0314472.2, filed Jun. 20, 2003. The entire teachingsof the above applications are incorporated herein by reference.

The invention relates to processes of making comb polymers from monomerscomprising alkoxy polyethers, such as polyalkylene glycol such as poly(ethylene glycol), or polytetrahydrofuran (PTHF). Such methods mayinclude the use of an initiator compound which comprises a moiety which,wizen attached to the comb polymer, is capable of binding to a proteinor polypeptide. The initiator compounds and finished comb polymers, andtheir uses, are also included within the invention.

The modification of proteins with polymers such as poly (ethyleneglycol), which is known by the abbreviation PEG, is well-known in theart. PEG-derivatives are manufactured, for example, by ShearwaterCorporation, Huntsville, Ala., USA, and Enzon, Inc., Bridgewater, N.J.,USA. Uses of PEG are reviewed in catalogues from both of thosecompanies, and indeed in the 2002 Enzon, Inc. Annual Report.

The attachment of PEG to proteins or polypeptides, known as PEGylationhas been found to have a number of benefits. Firstly, this reduces theantigenicity and immunogenicity of a molecule to which PEG is attached.PEG also produces markably improved circulating half-lives in viva dueto either evasion of renal clearance as a result of the polymerincreasing the apparent size of the molecule to above the glomerularfiltration limit, and/or through evasion of cellular clearancemechanisms. PEG can markably improve the solubility of proteins andpolypeptides to which it is attached, for example PEG has been found tobe soluble in many different solvents, ranging from water to manyorganic solvents such as toluene, methylene chloride, ethanol andacetone. An application of this has been to use PEG-modified antibodies,for example to phase partition target molecules or cells. PEGylation hasalso been found to enhance proteolytic resistance of the conjugatedprotein, and improve bioavailability via reduced losses at subcutaneousinjection sites. PEGylation also has been observed to reduce thetoxicity of the proteins or polypeptides to which it is attached,improve thermal and mechanical stability of the molecules and allow theimproved formulation into materials used for some slow releaseadministration strategies. These advantages are reviewed in, forexample, the articles by Chapman A. P. (Advanced Drug Delivery Reviews,Vol. 54 (2002), pages: 531-545). The chemistry of polypeptide andprotein PEGylation is further reviewed in the article by Roberts, M. J.,et al, (Advanced Drug Delivery Reviews, Vol. 54 (2002), pages 459-476),and the article by Kinstler, O., et al. (Advanced Drug Delivery Reviews,Vol. 54 (2002), pages 477.485).

A number of PEGylated drags are on the market, For example. PEG-INTRON™is an α-interferon product produced by Schering-Plough and Enzon, Inc.which is used to treat hepatitis C and cancer. Prothecan™ is aPEG-enhanced version of camptothecin, topoisomerase I inhibitor that iseffective against some cancers, PEGylated taxol and several enzyme-basedproducts have also been produced which show, for example, better uptakein tumours and reduced side-effects compared to non-PEGylated compounds.As discussed in the review by Roberts (Supra), polymers such as PEG maybe attached via a number of reactive amino acids on protein orpolypeptide molecules, including lysine, cysteine, histidine, arginine,aspartic acid, glutamic acid, serine, threonine, tyrosine, N-terminalamino groups and C-terminal carboxylic acid groups. In the ease ofglycoproteins, vicinal hydroxyl groups can be oxidised with periodate toform two reactive formyl moieties. A wide range of functional groups maybe attached to compounds such as PEG to allow them to attach to lysineamine groups and N-terminal amine groups. These include succinimidylsuccinate, hydroxysuccinamide and hydroxysuccinamide esters, aldehydederivatives such as propionaldehyde and acetaldehyde, propionate andbuterwate derivatives of succinimidyl, benzotriazole carbonate,ρ-nitrophenyl carbonate, trichlorophenyl carbonate andcarbonylimidazole. Compounds such as tresylate are known to bind toproteins via nucleophilic attack. There are also a number of compoundswhich can react with cysteine residues on proteins or polypeptides.These include maleimides, vinylsulphones, pyridyl sulphides andiodoacetamides. Furthermore, succinimidyl carbonate can also be used asa functionalised group to attach PEG or other polymers to alanine orhistidine amino acids within a protein or polypeptide. As alreadyindicated, the reaction of such functionalised groups is alreadywell-characterised as indicated in the articles by Roberts, Kinsler andChapman, and indeed as shown in, for example, the Shearwater Catalogue(2001).

The PEG currently on the market is usually in the form of long poly(ethylene glycol) polymers or branched or star-shaped poly (ethyleneglycols).

The Applicants have now identified that it is possible to produce combpolymers which allow the size of the polymer attached to biologicalsubstances, for example, proteins and polypeptides, nucleic acids (DNAand RNA), carbohydrates and fats, to be varied and to be controlled.This allows the possibility of producing a wide variety of differentpolymers for attaching to proteins and polypeptides, which may be variedin their size and hydrodynamic volume to vary the properties of thecompound to which the polymer is attached. For example, this may be usedto vary the stability, solubility, toxicity and/or drug retention timeof a drug which has been covalently attached to such co-polymers. Suchco-polymers are capable of being produced in, a controlled manner byso-called living radical polymerisation.

Living radical polymerisation is subject of International PatentApplication No. WO 97/47661. Supported polymerisation catalysts andspecific polymerisation initiators are also shown in WO 99/28352 and WO01/94424. Basically, the system uses a compound complexed with atransition metal. This compound is preferably an organodiimine, althoughone of the nitrogens of the diimine, is preferably not part of anaromatic ring (e.g. a 1,4-diaza-1,3-butadiene, a 2-pyridinecarbaldehydeimine, an oxazolidone or a quinoline carbaldehyde).

Living free radical systems, which involve the use of free radicalinitiators are also known, see for example WO 96/30421 and WO 97/18247.This is reviewed in Kamigaito, et al., Chem. Rev. (2001), Vol. 12, pages3689-3745.

A combination of the catalyst and the initiators has in the past beenused to polymerise un-saturated monomers, such as vinylic monomers. Theinventors have now realised that these systems may be used to producecomb polymers in a controlled manner. These comb polymers may have afunctional group attached to them via conventional chemistry. However,the inventors have also realised that the initiators used in livingradical polymerisation are attached to the comb polymer as a result ofthe reaction of the initiator with the monomers. This means that it ispossible to functionalise the comb polymer at the same time as producingthe co-polymer, by using a functionalised initiator.

Accordingly, the first aspect of the invention provides a method ofproducing a comb polymer comprising the steps of:

-   -   (a) Providing:        -   (i) a plurality of monomers which are linear, branched or            star-shaped, substituted or non-substituted, preferably            containing 2, especially from 3 to 10, carbon atoms, and            have an olefinically unsaturated moiety attached thereto,            the olefinically unsaturated moiety being capable of            undergoing addition polymerisation;        -   (ii) an initiator compound; the initiator compound            comprising as homolytically cleavable bond;        -   (iii) a catalyst capable of catalysing the polymerisation of            the monomer; and    -   (b) Causing the catalyst to catalyse, in combination with the        initiator, the polymerisation of a plurality of the monomers to        produce the comb polymer;        wherein the initiator compound (ii) comprises a moiety which,        when attached to the comb polymer, is capable of binding to a        biological substance.

The monomers in (i) are preferably alkoxy polyethers such as poly(alkylene glycol) or polytetrahydrofuran.

The comb polymer may have a moiety which, when attached to the combpolymer, is capable of binding e.g. a protein or polypeptide, attachedto it using conventional chemistry. However, as already indicated, it ispossible to produce initiator compounds which have that moiety attachedto them. Therefore, preferably the initiator compound comprises a moietywhich, when attached to a comb polymer, is capable of binding to abiological substance, such as a protein or polypeptide, nucleic acid(DNA or RNA), carbohydrates or fats.

Preferably, the poly (alkylene glycol) is a polymer of an alkyleneglycol containing from 2-10, especially at least 3, carbon atoms, mostpreferably poly (ethylene glycol), of (propylene glycol) or poly(butylene glycol). For example, poly (ethylene glycol) may be used.

In its most common form, this is a linear or branched polyetherterminated with hydroxyl groups. This is synthesised by anionic ringopening polymerisation of ethylene oxide initiated by nucleophilicattack of a hydroxide ion on the epoxide ring. It is also possible tomodify polyethylene glycol, for example by placing a monomethoxy groupon one end to produce monomethoxy PEG (mPEG). This is synthesised by anionic ring opening polymerisation initiated with methoxide ions and iscommercially available. However, trace amounts of water present in thereaction mixture causes the production of significant quantities of PEGwhich is terminated at both ends by hydroxy groups. This is undesirable,as the moiety capable of binding to proteins or peptides will thenattach to both ends of the polymer chain, which will cause unwantedcrosslinking of proteins in the body.

A method intended to minimise the production of this impurity is toinitiate the ring opening of ethylene oxide by nucleophilic attack of abenzoxy ion on the epoxide ring. In a similar manner to the aboveprocess, monobenzoxy PEG is produced, as well as the PEG chainterminated at both ends by hydroxy. This mixture is methylated,producing one chain terminated with BzO and OMe, and dimethoxy PEG.Hydrogenation of this mixture eliminates the benzoxy group to yield mPEGand dimethoxy PEG, Dimethoxy PEG remains present as an inert impurity.However, even using this process, the product obtained still contains5-10% of the unwanted dihydroxy PEG according to its certificate ofanalysis.

The process of the present invention yields a product which issubstantially 100% pure, eliminating substantially all of the dihydroxyPEG impurity, thus avoiding the disadvantages of the known processes,and removing the possibility of the cross-linking of proteins.

Branched and star-shaped polymers such as PEG are available from anumber of commercial sources, such as Enzon and Shearwater.Polytetrahydrofurans may also be obtained from commercial sources, suchas Aldrich (Gillingham, Dorset, UK.).

Preferably, the molecular weight of the PEGmethacrylate is 475, 1100,2080, 5000 or 20,000.

The polyalkylene glycol and polytetrahydrofuran comprises anolefinically unsaturated moiety, for example at the end of the polymerchain. This olefinically unsaturated moiety is capable of undergoingadditional polymerisation.

The olefinically unsaturated monomer may be a methacrylate, an acrylate,a styrene, methacrylonitrile or a diene such as butadiene.

Examples of olefinically unsaturated moieties that may be used includemethyl methacrylate, ethyl methacrylate, propyl methacrylate (allisomers), butyl methacrylate isomers), and other alkyl methacrylates;corresponding acrylates; also functionalised methacrylates and acrylatesincluding glycidyl methacrylate, trimethoxysilyl propyl methacrylate,allyl methacrylate, hydroxyethyl methacrylate, hydroxypropylmethacrylate, dialkylaminoalkyl methacrylates such as dimethylethylaminomethacrylate; fluoroalkyl (meth)acrylates; methacrylic acid, acrylicacid; fumaric acid (and esters), itaconic acid (and esters), maleicanhydride: styrene, α-methyl styrene; vinyl halides such as vinylchloride and vinyl fluoride; acrylonitrile, methacrylonitrile; glycerol;vinylidene halides of formula CH₂═C(Hal)₂ where each halogen isindependently Cl or F; optionally substituted butadienes of the formulaCH₂═C(R¹⁵) C(R¹⁵)═CH₂ where R¹⁵ is independently H. C1 to C10 alkyl, Cl,or F; sulphonic acids or derivatives thereof of formula CH₂═CHSO₂OMwherein M is Na, K, Li, N(R¹⁶)_(4i) where each R¹⁶ is independently H orC₁ to C₁₀ alkyl, COZ, ON, N(R¹⁶)₂ or SO₂OZ and Z is H, Li, Na, K orN(R¹⁴)₄; acrylamide or derivatives thereof of formula CH₂ CHCON(R¹⁶)₂and methacrylamide or derivative thereof of formula CH₂═C(CH₃)CON(R¹⁶)₂.

Mixtures of such monomers may be used.

Such unsaturated moieties may be attached, for example, at an end of thepolymer, by conventional chemistry. Alternatively, such monomers may beobtained commercially. For example, PEGacrylate, diacrylate,methacrylate and dimethacrylate are commercially available from Aldrich(Gillingham, Dorset, UK.).

The unsaturated moiety may be attached to the polyalkylene glycol orpolytetrahydrofuran by means of any suitable linkage groups, for examplevia a methyl ether linkage. Hence, it is possible to use poly (ethyleneglycol) methyl ether methacrylate (available from Aldrich Chemicals).One advantage of using the living radical polymerisation technique isthat commercially available compounds such as this, which havefree-radical inhibitors, such as hydroquinones, may be used withoutfurther purification. With conventional free-radical-based systems thepresence of a free-radical inhibitor will inhibit the additionpolymerisation reaction. This is not the case with living radicalpolymerisation.

The initiator compound may comprise a homolytically cleavable bond witha halogen atom. This may contain a bond that breaks without integralcharge formation on either atom by homolytic fission. As described in WO97/01589, WO 99/28352 and WO 01/94424, it is believed that truefree-radicals do not appear to be formed using some catalysts. It isbelieved that this occurs in a concerted fashion whereby the monomer isinserted into the bond without formation of a discrete free-radicalspecies in the system. That is, during propagation this results in theformation of a new carbon-carbon bond and a new carbon-halogen bondwithout free-radical formation. A free-radical which is an atom or groupof atoms having an unpaired valance electron and which is a separateentity without interactions, is not produced by the interaction of theinitiator compound with the monomer with which it interacts.

Suitable initiator compounds are described in, for example, WO 97/47661.However, it is preferable that the initiator compound also comprises amoiety which, when attached to the comb polymer, is capable of bindingto a protein or polypeptide. These moieties are known in the art, asindeed described in Roberts, et al. (Supra), Chapman (Supra) and, forexample, in the catalogues of Enzon and Shearwater.

The initiator may be a thioester or xanthate. These are used in socalled RAFT (Reversible Addition Fragmentation chain transfer and nitricoxide mediated polymerisation) and MADIX catalysation. The initiatorsand their reactions are described in WO 99/31144, WO 98/01478 and U.S.Pat. No. 6,053,705.

Preferably, the initiator compound (ii) is selected from

A-S—C(O)—R, A-S—C(S)—O—R, R—S—C(O)-A, R—S—C—(S)—O-A, when R is C₁ to C₂₀substituted or loon-substituted, straight chain, branched chain, cyclic,heterocyclic or aromatic alkyl;

where: X=a halide, especially Cl or Br,

-   -   A a moiety which, when attached to the comb polymer, is capable        of binding to a protein or polypeptide,    -   B is a linker and may or may not be present.

A is preferably selected from succinimidyl succinate, N-hydroxysuccimimide, succinimidyl propionate, succinimidyl butanoate,propionaldehyde, acetaldehyde, tresylate, triazine, vinylsulfone,benzotriazole carbonate, maleimide, pyridyl sulfide, iodoacetamide andsuccinimidyl carbonate.

The linker is preferably selected from a C₁ to C₂₀ substituted ornon-substituted, straight chain, branched chain cyclic, heterocyclic oraromatic alkyl group; —(CH₂Z)_(a) CH₂—, —CH₂ZCH₂—, —(CH₂CH₂Z)_(n)—R,—(CH₂CH(CH₃Z)_(n)—R, —(CH₂)_(b)—C(O)—NH—(CH₂)_(c)—,—(CH₂)_(a)—NH—C(O)—(CH₂)_(y)—, —N(H)₂—; —S—; —N—R; or —O—R; where R═C₁to C₂₀ substituted or non-substituted, straight chain, branched chaincyclic, heterocyclic or aromatic alkyl, Z is O or S, and n, a, b and care independently selectable integers between 1 and 10. Preferably, thelinker contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Mostpreferably, the linker is methyl, ethyl, propyl, butyl or pentyl.

Preferably, the moiety which is capable of reacting with the protein orpolypeptide has the formula:

where n=integer of 0 to 10

where m=integer of 0 to 10, Y is an aliphatic or aromatic moiety

where R′ is H, methyl, ethyl, propyl or but X is a halide, especially Clor Br.

Most preferably, the initiator (ii) has a formula:

where n is an integer of 0 to 10, and X is a halide, especially Cl orBr.

The initiator has a compound selected from

The catalyst may be capable of catalysing the polymerisation reaction byliving radical polymerisation (see e.g. WO 97/47661) or living freeradical polymerisation (see e.g. WO 96/30421, WO 97/18247 and KarnagaitoM., et al., Chem. Rev. (2001), Vol. 101 (12), pages 3689-3745).

Preferably the catalyst comprises a ligand which is any N—, O—, P— or S—containing compound which can coordinate in a δ-bond to a transitionmetal or any carbon-containing compound which can coordinate in a π-bondto the transition metal, such that direct bonds between the transitionmetal and growing polymer radicals ate not formed.

The catalyst may comprise a first compound

MY

-   -   where: M is a transition metal having an oxidation state which        is capable of being oxidised by one form a oxidation state,        -   Y is a mono, divalent or polyvalent counterion.

The catalyst may also be defined by the formula:

[ML_(m)]^(n+)A^(n−)

-   -   where:        -   M=a transition metal having an oxidation state which is            capable of being oxidised by one formal oxidation state,        -   L=an organodilmine where at least one of the nitrogens of            the diimine is not part of an aromatic ring,        -   A=anion,        -   n=integer of 1 to 3,        -   m=an integer of 1 to 2.

The metal on may be attached to a coordinating ligand, such as (CH₃CN)₄. Y may be chosen from Cl, Br, F, I, NO₃, PF₆, BF₄, SO₄, CN, SPh,SCN, SePh or inflate (CF₃SO₃). Copper (I) inflate may be used. This isavailable; in the form of a commercially available benzene complex(CF₃SO₃Cu)₂C₆H₆.

The especially preferred compound used is CuBr.

A may be F, Cl, Br, I, N, O₃, SO₄ or CuX₂ (where X is a halogen).

The transition metal may be selected from Cu⁺, Cu²⁺, Fe³⁺, Ru²⁺, Ru³⁺,Cr²⁺, Cr³⁺, Mo²⁺, Mo³⁺, W²⁺, W³⁺, Mn³⁺, Mn⁴⁺, Rh³⁺, Rh⁴⁺, Re²⁺, Re³⁺,Co⁺, Co²⁺, V²⁺, V³⁺, Zn⁺, Zn²⁺, Au⁺, Au²⁺, Ag⁺ and Ag²⁺.

Preferably the organodiimine has a formula selected from:

-   -   a 1,4-diaza-1,3-butadiene

-   -   a 2-pyridine carbaldehyde imine

-   -   an oxazolidone,

-   -   or a quinoline carbaldehyde

where R₁, R₂, R₁₀, R₁₁, R₁₂ and R₁₃ may be varied independently and R₁,R₂, R₁₀, R₁₁, R₁₂ and R₁₃ may be H, straight chain, branched chain orcyclic saturated alkyl, hydroxyalkyl, carboxyalkyl, aryl (such as phenylor phenyl substituted where substitution is as described for R₄ to R₉)CH₄Ar (where Ar=aryl or substituted aryl) or a halogen. Preferably R₁,R₂, R₁₀, R₁₁, R₁₂ and R₁₃ may be a C₁ to C₂₀ alkyl, hydroxyalkyl orcarboxyalkyl, in particular C₁ to C₄ alkyl, especially methyl or ethyl,n-propylisopropyl, n-butyl, sec-butyl, tert butyl, cyclohexyl,2-ethylhexyl, octyl decyl or lauryl.

Preferred ligands include:

-   -   R14=Hydrogen, C₁ to C₁₀ branched chain alkyl, carboxy- or        hydroxy-C₁ to C₁₀ alkyl.

Preferably the catalyst is

with CuBr

Preferably the organodiimine is N-(n-propyl)-2-pyridylmethanimine(NMPI), N-ethyl-2-pyridyl methanimine orN-(n-ethyl)-2-pyridylmethanimine.

Other catalysts are described in WO 96/30421 and WO 97/18247.

Preferably the catalyst comprises a bipyridine group, such as4,4′-di(5-nonyl)-2,2′-bipyridyl (dNbpy).

A plurality of different monomers as defined in part (i) of theinvention may be used. This allows the production of statisticalco-polymers.

Alternatively, or additionally, a block co-polymer may be produced byadditionally polymerising one or more different olefinically unsaturatedmonomers. For example, the olefinically unsaturated monomers may beselected from methyl methacrylate, ethyl methacrylate, propylmethacrylate all isomers), butyl methacrylate (all isomers), and otheralkyl methacrylates; corresponding acrylates; also functionalisedmethacrylates and acrylates including glycidyl methacrylate,trimethoxysilyl propyl methacrylate, allyl methacrylate, hydroxyethylmethacrylate, hydroxypropyl methacrylate, dialkylaminoalkylmethacrylates; fluoroalkyl (meth)acrylates; methacrylic acid, acrylicacid; fumaric acid (and esters), itaconic acid (and esters), maleicanhydride; styrene, α-methyl styrene; vinyl halides such as vinylchloride and vinyl fluoride; acrylonitrile, methacrylonitrile;vinylidene halides of formula CH₂═C(Hal)₂ where each halogen isindependently Cl or F; optionally substituted butadienes of the formulaCH₂═C(R¹⁵) C(R¹⁵)═CH₂ where R¹⁵ is independently H, C₁ to C₁₀ alkyl, Cl,or F; sulphonic acids or derivatives thereof of formula CH₂═CHSO₂OMwherein M is Na, K, Li, N(R¹⁶)₄ where each R is independently H or C1 toC10 alkyl, COZ, ON, N(R¹⁶)₂ or SO₂OZ and 2 is H, Li, Na, K or N(R¹⁶)₄;acrylamide or derivatives thereof of formula CH₂═CHCON(R¹⁶)₂ andmethacrylamide or derivative thereof of formula CH₂═C(CH₃)CON(R¹⁶)₂.

The monomers may be polymerised prior to or after the polymerisation ofthe monomers as defined in part (I) of the invention.

The polymerisation reaction may be reactive in a number of differentsolvents, such as hydrophobic or hydrophilic solvents. These includewater, propionitrile, hexane, heptane, dimethoxyethane, diethoxyethanetetrahydrofuran, ethylacetate, diethylether, N,N-dimethylformamide,anisole, acetonitrile, diphenylether, methylisobutyrate, butan-2-one,toluene and xylene.

The reaction temperature may be carried out from −20 to greater than200° C., especially +5 to 130° C. WO 97/47661, for example, showsexamples of living radical polymerisation and the typical conditionsthat may be used.

Preferably, the ratio of organodiimine:transition metal is 0.01 to 1000,preferably 0.1 to 10, and transition metal ion (as MY):initiator is0.0001 to 1000, preferably 0.1 to 10, where the degree of polymerisationis controlled by the ratio of monomer to initiator. All ratios are givenas weight weight. Preferably the components are the catalyst of formula:[ML_(m)]^(n+)A^(n−) (defined above) are at a ratio of catalyst;initiator of 3:1 to 1:100.

Preferably, the amount of diimine metal used in the system is between100:1 and 1:1, preferably 5:1 to 1:1, more preferably 3:1 to 1:1, byweight.

Preferably the concentration of monomer in a solvent used is 100%-1%,preferably 100%-5%, vol.:vol.

Preferred ratios of initiator to catalyst or 1:100-100:1, typically 1:1.

Preferred ratios of monomer:initiator are 1:1 to 10,000:1, especially5:1 to 100:1.

The reaction may be undertaken under an inert atmosphere such asnitrogen or argon, and may be carried out in suspension, emulsion,mini-emulsion or in a dispersion.

Preferably, the catalyst is a supported catalyst, that is at least apart of the catalyst is attached to a support. Such supported catalystsare shown in, for example, WO 99/28352.

The support may be inorganic, such as silica, especially silica gel.Alternatively, the support may be organic, especially an organicpolymer, such as a cross-linked organic polymer, including poly(styrene-w-dinylbertzone). The support may be in the form of beads. Theadvantage of using a supported catalyst is that it allows the catalystto be removed from the system and recycled/reused.

The comb polymer may incorporate a fluorescently-labelled monomer. Forexample, the method may additionally comprise a step of copolymerisingor block polymerising with at least one fluorescently-labelled monomercapable of undergoing addition polymerisation. This can be carried outsimply by using a monomer which has a fluorescent moiety, such asfluorescein, or coumarin, attached to an olefinically unsaturatedmoiety. The olefinically unsaturated moiety may be selected from thoseunsaturated moieties defined above.

Preferably, the fluorescent label is coumarin, especially coumarin 343.Coumarin is particularly advantageous because it allows the comb polymerto be used to attach to proteins and the attachment of the proteins tobe visualised using a confocal microscope. This allows, for example, thedetection of individual proteins or indeed the visualisation of wholebacterial or other cells. Indeed, initial results have indicated thatbacterial cells can be readily visualised, using a comb polymeraccording to the invention, to attach to E. coli and Streptomyces cells.

A further aspect of the invention provides initiator compounds capableof being used in a living radical polymerisation reaction comprising amoiety which, when attached to a polymer, is capable of binding to aprotein or polypeptide. Initiators for use in a living radicalpolymerisation reaction having the following formulae are also provided:

A-S—C(O)—R, A-S—C(S)—O—R, R—S—C(O)-A, R—S—C(S)O-A, where R is C₁ to C₂₀substituted or non-substituted, straight chain, branched chain, cyclic,heterocyclic or aromatic alkyl;

-   -   where:        -   X=a halide, especially Cl or Br,        -   A=a moiety which, when attached to the comb polymer, is            capable of binding to a protein or polypeptide,        -   B is a linker and may or may not be present.

Preferably, A is selected from succinimidyl succinate, N-hydroxysuccimimide, succinimidyl propionate, succinimidyl batanoate,propionaldehyde, acetaldehyde, tresylate, triazine, vinyl sulfone,benzotriazole carbonate, maleimide, pyridyl sulfide, iodoacetamide andsuccinimidyl carbonate.

Preferably, the linker is selected from a C₁ to C₂₀ substituted ornon-substituted, straight chain, branched Chain cyclic, heterocyclic oraromatic alkyl group; —(CH₂Z)_(a) CH₂—, —CH₂ZCH₂—, —(CH₂CH₂Z)_(n)—R,—(CH₂CH(CH₃Z)_(n)—R, —(CH₂)_(b)—C(O)—NH—(CH₂)_(c)—,—(CH₂)_(a)—NH—C(O)—(CH₂)_(y)—, —N(R)₂—; —S—; —N—R; or —O—R; where R=C₁to C₂₀ substituted or non-substituted, straight chain, branched chaincyclic, heterocyclic or aromatic alkyl, Z is O or S, and n, a, b and care independently selectable integers between 1 and 10,

Preferably the moiety capable of reacting with a protein or polypeptidehas a formula:

where n=integer of 0 to 10

where m=integer of 0 to 10, Y is an aliphatic or aromatic moiety

where R′ is H, methyl, ethyl, propyl or butyl, X is a halide, especiallyCl or Br.

Preferably the initiator has a formula of:

where n is an integer of 0 to 10, and X is a halide, especially Cl orBr.

The initiator especially has the formula:

The terminal amine group may be protected by any suitable protectinggroup, such as BOC. Deprotection is achieved by addition of acid, suchas trifluoroacetic acid. Alternatively a furan intermediate may beproduced which can then be converted to maleimide.

Under normal conditions, the aldehyde-based initiators will tend toreact non selectively with proteins, i.e, they will react substantiallyequally with both terminal nitrogen atoms and, for example, a lysine NH₂group, if the reaction conditions are not controlled. However, under theright reaction pKa for the particular aldehyde chosen, the aldehyde canbe controlled to specifically target the terminal nitrogen. A furtheraspect of the invention provides comb polymers capable of bindingprotein or polypeptide obtainable by a method of the invention.

A further aspect provides a comb polymer having a general formula:

A-(D)_(d)-(E)_(e)-(F)_(f)

where:

-   -   A may or may not be present, and where present is a moiety        capable of binding to a protein or a polypeptide,    -   D, where present, is obtainable by additional polymerisation of        one or more olefinically unsaturated monomers which are not as        defined in E,    -   E is obtainable by additional polymerisation of a plurality of        monomers which are linear, branched, or star-shaped, substituted        or non-substituted, and have an olefinically unsaturated moiety,    -   F, where present, is obtainable by additional polymerisation of        one or more olefinically unsaturated monomers which are not as        defined in E,    -   d and f are an integer between 0 and 500, especially 0 to 300 or        0 to 100,    -   e is an integer of 0 to 1000, especially 0 to 10, 50, 100, 200,        300, 400, 500, 600, 700, 800 or 900    -   and wherein when A is present, at least one of D, F and F is        present.

Preferred monomers used to obtain E are poly (alkylene glycol) orpolytetrahydrofuran.

This includes both functionalised comb polymer and non-functionalisedcomb polymer, where the moiety capable of attaching to a protein orpolypeptide may be attached later by other chemistry.

Preferably the comb polymer has an average total, molecular weight of2,000-80,000, especially 20,000-40,000.

Examples of preferred comb polymers, obtainable according to the processof the invention, are

These polymers can be used either directly to react with usefulbiomolecules or converted simply into new macromolecules that will reactwith useful biomolecules.

The comb polymer may be fluorescently labelled, especially with acoumarin. A still further aspect of the invention provides a method ofattaching a polymer to a compound comprising reacting a comb polymeraccording to the invention with said compound. The compound may be aprotein or polypeptide or may indeed be any compound having a suitablefree thiol or free amine group, depending on the initiator used. Suchcompounds include amines, such as benzylamines and ethylenediamine,amino acids and carbohydrates such as sugars.

Preferably such compounds are biologically-active compounds, such asdrugs. The combination of such compounds in combination with apharmaceutically acceptable carrier are also provided. The compounds mayinclude cancer chemotherapeutic agents, antibiotics, anti-fungal and/orimmunosuppressants.

For example, FIGS. 23 and 24 show HPLC traces and SDS-PAGE for thereaction of lysozyme with a polymer prepared according to the invention.These figures clearly illustrate the progress of the reaction as thepolymer selectively conjugates to only one of lysozymes seven aminogroups.

A still further aspect of the invention provides a method offluorescently labelling a compound, virus, microorganism or cellcomprising the step of reacting the compound, virus, microorganism orcell with a fluorescently labelled comb polymer according to theinvention. The use of a comb polymer as a fluorescent label is alsoprovided.

The fluorescently labelled comb polymer may be used to attach antibodieswhich in turn may be used to selectively bind to pre-defined antigens.This allows the selective labelling of the compounds to take place.

Methods of producing such antibodies are well-known in the art andindeed monoclonal antibodies may be produced by the well-knownKohler-Milstein method.

Previously, when polymers have been used to bind to proteins, they havehad to be of a low molecular weight, as a polymer with a molecularweight of e.g. 20,000 could not be excreted from the body by the liver.To combat this problem, four polymers of approximately 5,000 molecularweight each were bound to the protein, and eventually excreted withoutproblem. An advantage that is provided by the comb polymers of theinvention is that they can possess molecular weights of 20,000 and stillbe bound to the proteins without the problems of excretion found withconventional polymers. This is due to an ester linkage which is found ineach “finger” of the comb polymer. Preliminary results show that thisester linkage is readily hydrolysed by enzymes, causing the fingers togradually break off the main polymer backbone. This enables a 20,000molecular weight polymer to become smaller over time until it reaches amolecular weight which enables it to be excreted by the liver.Conventional chain polymers cannot offer this advantage but remain inthe bloodstream without being excreted.

Initial results indicate that the comb polymers of the invention arestable over weeks in at serum, but slowly break down in the mannerdetailed above.

The invention will now be described by way of example only withreference to the following examples:

FIG. 1 shows the evolution of molecular weight distribution andpolydispersity for the LRP (living radical polymerisation) of methylmethacrylate initiated by a N-hydroxysuccinimide (NHS) initiator.

FIG. 2 shows SEC curves for NHS functionalised poly (MMA), solid curve,and the produce (N-benzylamide functionalised poly (MMA), dashed curve).

FIG. 3. First order kinetic plot for the LRP of PEGMA initiated byNHS—Br, [PEGMA]_(o)/[CuBr]_(o)/[NHSBr]_(o)/[L]_(o)=10/1/1/2.1 in toluene(33% v/v) at 30° C.

FIG. 4. Evolution of the molecular weight distribution andpolydispersity for the LRP of PEGMA initiated by NHS—Br,[PEGMA]_(o)/[CuBr]_(o)/[NHSBr]_(o)/[L]_(o)=10/1/1/2.1 in toluene (33%v/v) at 30° C.

FIG. 5. Evolution of the molecular weight distribution andpolydispeersity for the LRP of MPEG(395)MA initiated using initiator 8,[MPEG(395)MA]₀[CuBr]₀/[NHSBr]₀=10/1/1/2 in tuluene (50% v/v) at 30° C.

FIG. 6. Selected region (2.7-4.3 ppm) of the ¹H NMR spectrum of a NHSester functionalised poly(MPEG(395)MA) prepared from initiator 8(M_(n)=6400 g·mol⁻¹, M_(w)/M_(n)=1.09).

FIG. 7. First order kinetic plot for the LRP of MPEG(395)MA usinginitiator 7, [MPEG(395)MA]₀/[CuBr]₀/[NHSBr]₀/[Propyl Ligand]₀=10/1/1/2ub toluene (50% v/v) at 30° C.

FIG. 8. Evolution of the molecular weight distribution andpolydispersity for the LRP of MPEG(395)MA using initiator 7,[MPEG(395)MA]₀/[CuBr]₀/[NHSBr]₀/[Propyl Ligand]₀=10/1/1/2 in toluene at30° C.

FIG. 9. Rate plot for TMM-LRP of MPEG(1000)MA initiator 12,[monomer]:[initiator]:[CuCl]:[L]=5:1:2:2, T=70° C.

FIG. 10. Dependence of M_(n) on conversion for MPEG(1000)MA initiator12, [monomer]:[initiator]:[CuCl]:[L]=5:1:1:2, T 70° C.

FIG. 11. Rate plot thr TMM-LRP of MPEG(1000)MA initiator 12,[monomer]:[initiator]:[CuBr]:[L]=20:1:1:2, T=50° C.

FIG. 12. Dependence of M_(n) on conversion for TMM-LRP of MPEG(1000)MAinitiator 12, [monomer]:[initiator]:[CuBr]:[L]=20:1:1:2, T=50° C.

FIG. 13. Rate plot for TMM-LRP of MPEG(395)MA using initiator 14,[monomer]:[initiator]:[CuBr]:[L]=6:1:1:2.

FIG. 14. Dependence of M_(n) on conversion for TMM-LRP of MPEG(395)MAusing initiator 14, [monomer]:[initiator]:[CuBr]:[L]=6:1:1:2.

FIG. 15. Rate plot for TMM-LRP of MPEG(395)MA using initiator 14,[monomer]:[initiator]:[CuBr]:[L]=28:1:1:2, T 40° C.

FIG. 14. Dependence of M_(n) on conversion for TMM-LRP of MPEG(395)MA,using initiator 14, [monomer]:[initiator]:[CuBr]:[L]=6:1:1:2, T=40° C.

FIG. 17. Rate plot for TMM-LRP of MPEG(395)MA using initiator 14,[monomer]:[initiator]:[CuBr]: [L]=28:1:1:2, T=60° C.

FIG. 18. Dependence of M_(n) on conversion for TMM-LRP of MPEG(395)MAusing initiator 14, [monomer]:[initiator]=6:1:1:2, T=60° C.

FIG. 19. Online ¹H NMR experiment: Rate plot for TMM-LRP of NWEG (395)MAusing initiator 14, [monomer]: [initiator]:[CuBr]:[L]=10:1:1:2. T=40° C.

FIG. 20. Rate plot for TMM-LRP of MPEG(395)MA using initiator 15,[monomer]:[initiator]:[CuBr]:[L]=8:1:1:2. T=30° C.

FIG. 21. Dependence of M_(n) on conversion for TMM-LRP of MPEG(395)MAusing initiator 15, [monomer]:[initiator]:[CuBr]:[L]=8:1:1:2, T=30° C.

FIG. 22. Kinetic plot for the hydrolysis of N-succinimidyl terminatedpoly(MPEG(395)MA initiated by 8 in different buffers.

FIG. 23. HPLC traces for the reaction of succinimide terminatedpoly(MPEG(395)MA) prepared from initiator 8 (M_(n)=6400 g·mol⁻¹,M_(w)/M_(n)=1.11) with Lysozyme ([polymer]/[lysozyme] 20:1).

FIG. 24. SDS-PAGE for the conjugation of lysozyme with succinimideterminated poly(MPEG(395)MA) prepared from initiator 8 (M_(n)=6400g·mol⁻¹, M_(w)/M_(n)=1.11) (20 equivalents).

FIG. 25. HPLC traces for the reaction of succinimide terminatedpoly(MPEG(395)MA) prepared from initiator 8 (M_(n)=6400 g·mol⁻¹,M_(w)/M_(n)=1.11) with Lysozyme ([polymer]/[lysozyme] 5:1).

FIG. 26. HPLC traces for the reaction of succinimide terminatedpoly(MPEG(395)MA) prepared from initiator 8 (M_(n)=6400 g·mol⁻¹,M_(w)/M_(n)=1.11) with Lysozyme ([polymer]/[lysozyme] 2:1).

FIG. 27. Comparison of the HPLC traces of various conjugates of lysozymeobtained with different ratios of polymer/lysozyme using succinimideterminated poly(MPEG(395)MA) prepared from initiator 8.

FIG. 28. Kinetic plot for the hydrolysis of the succinimide end group ofpoly(MPEG(395)MA) polymer initiated by 7 in different buffers.

FIG. 29. ¹H NMR spectrum of a NHS eater functionalised (initiator 7)poly(MPEG(395)MA) (M_(n), 2700 g·mol⁻¹, M_(w)/M_(n)=1.12).

FIG. 30. ¹H NMR spectrum of a N-benzylamide functionalisedpoly(MPEG(395)MA) (M_(n)=2880 g·mol⁻¹, M_(w)/M_(n)=1.15).

FIG. 31. HPLC traces for the reaction of poly(MPEG(395)MA) prepared frominitiator 7 (M_(n)=2700 g·mol⁻¹, M_(w)/M_(n)=1.12) with lysozyme([polymer]/[lysozyme] 30:1).

FIG. 32. SDS-PAGE for the conjugation of poly(MPEG(395)MA) prepared frominitiator 7 with lysozyme at different reaction time and different ratiopolymer/protein (a) 5/1, (b) 10/1 and (c) 30/1.

FIG. 33. SEC-HPLC chromatography of the conjugation reaction of Lysozymewith the aldehyde-terminated polymer (M_(n)˜22,000 PDI 1.09).

FIG. 34. Retro-Diels-Alder reaction: (=“initiator” and

=maleimido signals) a) t=0; b) t=3.5 h; c) t=7 h.

SYNTHESIS OF N-[2-(2′-BROMO-2′-METHYLPROPIONYLOXY)-ETHYL]PHTHALIMIDE, 6

N-(2-hydroxyethyl)phthalimide (Aldrich, 99%) (19.12 g, 0.1 mol) wasdissolved in anhydrous THF (350 mL) with triethylamine (28.1 mL, 0.2mot) under nitrogen in a 500 mL round-bottomed flask equipped with amagnetic stirrer. The flask was cooled to 0° C. with an ice bath beforethe dropwise addition of 2-bromoisobutyryl bromide (13.9 mL, 0.11 mol).The mixture was stirred for 45 minutes and allowed to reach roomtemperature. Subsequently the reaction mixture was poured into an excessof cold water and extracted with diethyl ether (3×50 mL). The organiclayer was washed with a saturated aqueous solution of Na₂CO₃ (3×50 mL),acidified water (pH=4.5, 3×50 mL), and again the saturated aqueoussolution of Na₂CO₃ (3×50 mL). The organic layer was dried over anhydrousMgSO₄ and filtered. Finally the solvent was removed under reducedpressure by using the rotary evaporator in order to isolate the titlecompound (30.6 g, yield 90%) as a yellowish solid.

m.p. 63-65° C., IR (solid, ATR cell) ν (cm⁻¹) 1774 (C_(cycl)═O), 1705(C═O); ¹H NMR (CDCl₃, 298 K. 300 MHz) δ 1.81 (s, 6H, C(CH₃)₂Br), 3.95(t, 2H, J=5.3 Hz, CH₂N), 4.35 (t, 2H, J=5.4 Hz, CH₂O), 7.67 (m, 2H, CHAr), 7.78 (m, 2H, CH Ar). ¹³C NMR (CDCl₃, 298 K, 75 MHz) δ 31.00 (2C,C(CH₃)₂Br), 37.12 (1C, CH₂N), 55.92 (1C, C(CH₃)₂Br) 63.42 (1C, CH₂O),123.78 (2C, CH Ar), 132.35 (1C, C Ar), 133.54 (2C, CH Ar), 168.40 (2C,C_(cycl)═O), 171.87 (1C, C═O).

Synthesis of N-(2-bromo-2-methylpropionyloxy)succinimide, 7

This was prepared from N-hydroxysuccinimide (NHS) using a similarprocedure to that given above for the synthesis of compound 6. Thesolvent used in this case was anhydrous dichloromethane as NHS isinsoluble in THF. The title compound was obtained in 85% yield as awhite solid.

m.p. 72-74° C.; IR (solid, ATR cell) ν (cm⁻¹) 1772 (C_(cycl)═O), 1728(C═O); ¹H NMR (CDCl₃, 298 K, 300 MHz) δ 2.08 (s, 6H, C(CH₃)₂Br), 2.87(a, 4H, CH₂). ¹³C NMR (CDCl₃, 298 K 75 MHz) δ 26.03 (2C, CH), 31.09 (2C,C(CH₃)₂Br), 51.60 (1C, C(CH₃)₂Br), 167.89 (1C, C═O) 169.02 (2C,C_(cycl)═O), MS (+EI), (m/z) 266, 265, 156, 151, 149, 123, 121, 116,115, 91, 87, 70, 69. Anal, Calcd for C₈H₁₀NO₄Br: C=36.39; H=3.82;N=5.30, Br=30.26. Found: C=36.35; H=3.82; N=5.03; Br=30.17.

4-[(4-chloro-6-methoxy-1,3,5-triazin-2-11)amino]phenol, 4

A solution of 2,4-dichloro-6-methoxy-1,3,5-triazine²⁹ (9.00 g, 50.0mmol) in 100 mL of acetone was cooled to 0° C. and, under stirring,solid 4-aminophenol (5.46 g, 50.0 mmol was added in small portions overea. 2 min. The white suspension was then left to warm to ambienttemperature and stirred for further 1 h, whilst being neutralized with a2 M aqueous solution of Na₂CO₃. The mixture was then poured into 500 mLof ice/water and the resulting white precipitate was filtered and dried,to give 9.60 g (38.0 mmol, yield 76%) of4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenol that can be usedfor the next step without further purifications. An analytical samplewas obtained by flash chromatography (CC, SiO₂, petroleum ether/Et₂O1:1, R_(f)=0.14). The NMR analysis (d₆-DMSO) revealed the presence, insolution, of 2 rotational isomers (molar ratio 7:3).

m.p. 172° C. dec.; IR ν_((NH)) 3476 cm⁻¹. ν_((OH)) 3269 cm⁻¹.

Major isomer ¹H NMR (d₆-DMSO, 298K, 300 MHz) δ 3.94 (s, 3H, OCH₃); 6.79(d, J=8.8 Hz, 2H, CH Ar), 7.48 (d, J=8.8 Hz, 2H, CH Ar), 9.40 (s, 1H,OH), 10.46 (s, 1H, NH); ¹³C{¹H}NMR (d₆-DMSO, 298K, 75 MHz) δ 55.52 (1C,OCH₃); 115.46 (2C, CH Ar), 123.91 (2C, CH Ar), 129.49 (1C, C Ar), 154.44(1C, C Ar), 164.81 (1C, C Ar), 169.57 (1C, C Ar), 171.23 (1C, C Ar).

Minor isomer ¹H NMR (d₆-DMSO, 298K, 300 MHz) δ 3.96 (s, 3H, OCH₃); 6.79(d, J=8.9 Hz, 2H, CH Ar), 7.39 (d, J=8.9 Hz, 2H, CH Ar), 9.42 (bs, 1H,OH), 10.10.32 (s, 1H, NH); ¹³C{¹H} NMR (d₆-DMSO, 298 K, 75 MHz) δ 55.10(1C, OCH₃); 115.46 (2C, CH Ar), 123.03 (2C, CH Ar), 129.26 (1C, C Ar),154.76 (1C, C Ar), 165.20 (1C, C Ar), 170.48 (1C, C Ar), 170.64 (1C, CAr); Anal. Calcd for C₁₀H₉CN₄O₂: C^(=47.54), H=3.59, N=22.18, Cl=14.03.Found: C=47.57, H=3.55, N=22.10, Cl=14.8.

14-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenyl2-bromo-2-methylpropionate, 5

A solution of 2-bromoisobutyryl bromide (1.0 mL, 7.90 mmol) in 20 mL ofTHF was added dropwise to a solution of4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenol (1.9 g, 7.52mmol) and triethylamine in 100 mL of THF, at −10° C. During the addition(ca. 15 min) precipitation of triethylammonium bromide was observed. Thereaction was monitored by TLC (SiO₂, petroleum ether/Et₂O 1:1,4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenol (startingmaterial) R_(f)=0.14;4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenyl2-bromo-2-methylpropionate (final product) R_(f)=0.26). After 1.5 h thewhite suspension was poured into a conical flask containing 150 mL ofEt₂O and the ammonium salt removed by filtration on a sintered glassfrit. The solvent was then evaporated at reduced pressure to give awhite crude residue that was suspended in 10 mL of pentane and filtered.We obtained 2.56 g (6.37 mmol, yield 85%) of4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenyl2-bromo-2-methylpropionate as a white solid. The ¹H NMR analysis(d₆-DMSO) revealed the presence, in solution, of 2 rotational isomers(molar ratio 7:3).

m.p. 107-108° C.; IR ν_((NH)) 3365 cm⁻¹. ν_((C═O)) 1747 cm⁻¹.

Major isomer: ¹H NMR (d₆-DMSO, 298 K, 400 MHz) δ 2.05 (s, 6H,C(CH₃)₂Br), 3.96 (s, 3H, OCH₃), 7.17 (d, J=8.9 Hz, 2H, CH Ar), 7.77 (d,J=8.9 Hz, 2H, CH Ar), 10.78 (s, 1H, NH); ¹³C{¹H} NMR (d₆-DMSO, 298 K,100.6 MHz) δ 30.42 (2C, CH₃), 55.75 (bs, 1C, OCH₃), 57.29 (1C,C(CH₃)₂Br), 121.96 (2C, CH Ar), 122.12 (2C, CH Ar), 136.29 (1C, C Ar),146.61 (bs, 1C, C Ar), 165.10 (bs, 1C, C Ar), 169.89 (bs, 1C, C Ar),170.16 (1C, C═O), 171.33 (bs, 1C, C Ar).

Minor isomer: ¹H NMR (d₆-DMSO, 298 K, 400 MHz) δ 2.05 (s, 6H,C(CH₃)₂Br), 3.96 (s, 3H, OCH₃), 7.17 (d, J=8.9 Hz, 2H, CH Ar), 7.69 (d,J=8.9 Hz, 2H, CH Ar), 10.66 (s, 1H, NH); ¹³C(¹H) NMR (d₆-DMSO, 298 K,100.6 MHz) δ 30.42 (2C, CH₃), 55.75 (bs, 1C, OCH₃), 57.29 (1C,C(CH₃)₂Br), 121.96 (2C, CH Ar), 122.73 (2C, CH Ar), 136.29 (1C, C Ar),146.61 (bs, 1C, C Ar), 165.10 (bs, 1C, C Ar), 169.89 (bs, 1C, C Ar),170.16 (1C, C═O), 171.33 (bs, 1C, C Ar).

Typical Polymerisation of MMA.

CuBr (0.134 g, 0.934 mmol) was placed in an oven-dried Schlenk tube. Thetube was fitted with a rubber septum, evacuated and flushed with dry N₂three times. Methyl methacrylate (10 mL, 93.4 mmol) and xylene (20 mL)were transferred to the tube via degassed syringe. The mixture wasstirred rapidly under nitrogen and N-(n-propyl)-2-pyridylmethanimine(NMPI) (0.408 g, 1.86 mmol) was added which imparted a deep red/browncolour to the solution. Appropriate initiator (0.934 mmol) was added andthe resulting solution was degassed by three freeze-pump-thaw cycles.The resulting mixture was placed in a thermostatically controlled oilbath at 90° C. Samples were taken periodically for conversion andmolecular weight analysis. Conversion was measured by gravimetry bydrying to constant weight in a vacuum oven at 70° C. The catalyst wasremoved from the samples by passing through a column of activated basicalumina prior to SEC. (see FIG. 1).

TABLE 1 Polymerisation of MMA in Xylene Solution (33% v/v) at 90° C.[MMA]/ Cu(I)Br] [Cu(II)/ Br₂/[NMPI]/ Time M_(n) Conv Kp[Pol*]^(a) *Initiator [Initiator] Min G mol⁻¹ PDi % 10⁴ s⁻¹ 5 100/1/0/2/1^(c) 4808200 1.14 66 6 100/1/0/1/2.1 600 8900 1.20 75 0.047  (9000^(b)) 737/1/0/1/2.1 138 5800 1.05 89 0.32  (5800^(b)) 7 60/0.95/0.05/ 2880 32001.04 37 0.22 1/2.1  (3100^(b)) EiBr 100/1/0/2/1^(d) 2880 2500 1.16 71 (2400^(b)) ^(a)k_(p)[Pol*] = rate constant of propagation × [activepropagating polymer chains] from first order kinetic plot.^(b)determined by the ¹H NMR peak intensity ratio on a Bruker DPX 300MHz ^(c)N-(n-octyl)-2-pyridylmethanimine used as the ligand ^(d)10 mole% HEMA/90 mole % MMA

Typical Polymerisation of Styrene.

CuBr (0.055 g, 0.38 mmol) was placed in an oven dried Schlenk tube. Thetube was fitted with a rubber septum, evacuated and flushed three timeswith dry N₂. Styrene (10 mL, 96 mmol) was transferred, to the tube viadegassed syringe. The mixture was stirred rapidly under nitrogen and4,4′-di(5-nonyl)-2,2′-bipyridyl (dNbpy) (0.314 g, 0.768 mmol) was added,imparting a deep red/brown colour to the solution. Initiator 7 (0.035 g,0.048 mmol, 0.192 mmol of initiating sites) was added and the resultingsolution was degassed by three freeze-pump-thaw cycles. The resultingmixture was placed in a thermostatically controlled oil bath 110° C. for4.5 hours. The catalyst was removed from the samples by passing througha column of activated basic alumina prior to SEC.

Kinetic Studies for Initiators 6 and 1%

Samples were removed periodically using degassed syringes and quenchedin liquid nitrogen for conversion and molecular weight analysis.Conversion was determined by NMR on a Bruker DPX 300. For Living RadicalPolymerisation initiated by 6, samples were passed over a basic aluminacolumn and then filtered in a syringe equipped with a 0.22 μmhydrophobic filter prior to molecular weight studies. In the ease of LRPinitiated by 7, molecular weight was determined by diluting the samplewith THF and letting it settle overnight to precipitate the catalystresidues. The upper liquid was then filtered with a 0.22 μm hydrophobicfilter. This method was chosen for N-hydroxysuccinimide-functionalisedpolymers as these polymers could not be passed over basic alumina.

Synthesis of a N-Benzylamide Functionalised Poly(MMA)

Benzylamine was added to a solution of N-hydroxysuccinimide terminatedpoly(methyl methacrylate) in anhydrous THF.N-hydroxysuccinimide-terminated poly(methyl methacrylate) (M_(n)=3200 gmol⁻¹, PDI=1.06) (1.00 g, 0,313 mmol) and three equivalents ofbenzylamine (0,100 mL, 0,938 mmol) were dissolved in 10 mL of dry THF ina dry Schlenk and stirred at 50° C. for 3 days under nitrogen. Afterreaction, the polymer was precipitated in cold petroleum ether (see FIG.2).

This shows that N-benzylamide functional groups may be added and can beused to reach with free amide groups of the sort found in proteins.

N-Hydroxysuccinimide Initiator (7) (NHS—Br) Reagents.

Poly(ethylene glycol) methyl ether methacrylate (M_(n)=ca 475, Aldrich,99%) and anhydrous toluene was degassed by bubbling with dry nitrogenfor 30 minutes before use. The ligand N-(n-propyl)-2-pyridylmethaniminewas prepared as described previously¹. Copper(I) bromide (Avocado, 98%)was purified as necessary by a method based on that of Keller andWyooft². Other reagents were all commercial products and used withoutfurther purification.

Typical Procedure.

Polymerizations were carried out at 30° C. mediated by copper(I)bromide/N-(n-propyl)-2-pyridylmethanimine. A typical polymerizationrecipe is based on 33% v/v monomer in toluene. The ratio ofinitiator/Cu(I)Br/ligand is 1/1/2.1 on a molar basis. A dry Schlenk tubewas charged with Cu(I)Br (0.3099 g, 2.16×10⁻³ mol), NHS—Br (7) (0.5704g, 2.16×10⁻³ mol) and a magnetic bar prior to being deoxygenated bycycling between nitrogen and vacuum three times. To the flask was thenadded PEGMA (10 ml, 2.27×10⁻² mol) and toluene (20 ml). The mixture wasimmediately subjected to three freeze-pump-thaw degassing cycles.Finally N-(n-propyl)-2-pyridylmethanimine (0.707 ml, 4.54×10⁻³ mol) wasadded and the flask was placed in an oil bath thermostatted at 30° C.

Kinetic Studies.

Samples were removed periodically using degassed syringes and quenchedin liquid nitrogen for conversion and molecular weight analysis.Conversion was determined by NMR on a Brüker DPX 300 MHz. Molecularweight was determined by diluting the sample with toluene and allowingit to settle down overnight to remove the copper complexes. The upperliquid was then filtered with a 0.22 μm hydrophobic filter. This methodwas chosen because of the difficulty encountered to pass the polymerover a basic alumina column. Number average molecular weights (M_(n))were determined by Size Exclusion Chromatography (SEC) in a systemfitted with a 5 mm guard column, two Polymer Labs mixed E columns, adifferential refractive index detector, and an auto sampler. The systemwas elided with THP at a rate of 1 mL/min. Toluene was used as the flowmarker.

Purification.

N-hydroxysuccinimide functionalised poly(PEGMA) were purified by twoconsecutive purifications from a Toluene solution in diethyl ether.

TABLE 1 Kinetic data for the polymerisation of PEGMA initiated by NHS-Brin toluene solution (33% v/v) at 30° C. ([PEGMA]₀/[CuBr]₀/[NHSBr]₀/[L]₀= 10/1/1/2.1). Time Conversion M_(n,exp) M_(n,theo) ^(b) (h) (%) (g,mol⁻¹) M_(w)/M_(n) ^(a) (g, mol⁻¹) 1 8.9 2350 1.10 450 2 18.4 2860 1.26920 3 27.1 3100 1.20 1360 4 34.7 3600 1.13 1730 17 80.8 5670 1.06 4040^(a) determined by SEC analysis calibrated with Poly(MMA) standards -THF. ^(b) M_(n,theo) = ([M]₀/[I]₀ × M.W._(MMA) × Conv.)/100.

TABLE 2 Characterisation of Poly(PEGMA) prepared by LRP Kp[Pol*] ^(a)M_(n,exp) ^(b) M_(n,theo) ^(b) (h⁻¹) (g, mol⁻¹) M_(w)/M_(n) (g, mol⁻¹)NHS-Poly(PEGMA) 0.096 6200 1.05 4040 ^(a) Kp[Pol*] = rate constant ofpropagation. ^(b) determined by SEC calibrated with Poly(MMA)standards - THF (stabilised with topanol). ^(b) M_(n,theo) = ([M]₀/[I]₀× M.W._(MMA) × Conv.)/100.

REFERENCES

-   (a) D. M. Haddletori, M. C. Crossman, B. H. Dana, D, J.    Duncalf, A. M. Henning, D. Kukulj and A. J. Shooter, Macromolecules,    1999, 32, 2110.-   (b) R. N. Keller and W. D. Wycoff, Inorg. Synth., 1947, 2, 1.

Polymerisation of Methoxypolyethyleneglycol Methacrylate (2080) Usingthe Initiator Thrived from N-Hydroxy Succinimide[PEG]/[I]/[Cu]/[L]=19.2/1/1/2 in 80% Toluene Solution (AJ U2-27a)@30° C.

N-hydroxy succinimide initiator, (0.05 g, 0,189 mmol), Cu(I)Br (0.027 g,0.189 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG)(average molecular weight=2080, 7.55 g, 3.63 mmol), and a magneticfollower were placed in an oven dried Schlenk tube. The Schlenk tube wasevacuated and flushed with dry nitrogen three times. Deoxygenatedtoluene (28 mL) was added to the Schlenk tube. The resulting solutionwas deoxygenated via three freeze pump thaw cycles and then degassedN-ethyl-2-pyridylimethanimine (0.05 g, 0.38 mmol) was added. Thereaction was placed in a thermostatically controlled oil bath at 30° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis. Conversion was followed by ¹H NMR spectrometry andmolecular weight analysis by SEC.

The polymer was purified by the dropwise addition of the reactionsolution to a vigorously stirred solution of diethyl ether (400 mL). Theresulting white powder was filtered, dissolved in toluene (20 mL) andprecipitated in diethyl ether (400 mL). This procedure was repeatedthree times.

TABLE 1 Data for the polymerization of methoxypolyethyleneglycolmethacrylate (2080) with an initiator derived from N-hydroxy succinimideat 30° C. in 80% toluene solution. Sample Time/minutes Conversion^(a)/%Mn^(b) PDi^(b) 89 4 3380 1.04 291 9 9820 1.09 901 17 10030 1.07 1369 2311080 1.07 2760 26 12610 1.07 3965 28 14830 1.04 ^(a)Conversion wasdetermined using 1H NW. ^(b)Molecular mass determined by SEC using PMMAstandards.

Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycolmethacrylate) was freeze dried prior to use to remove all water.

[PEG]/[I]/[Cu]/[L]=19.2/1/1/2 in 80% Toluene Solution (AJ U2-27b) @50°C.

N-hydroxy succinimide initiator, (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g,0.189 mmol, 1 eq) and methoxypolyethyleneglyeol methacrylate (PEG)(average molecular weight=2080, 7.55 g, 3.63 mmol), and a magneticfollower were placed in an oven dried Schlenk tube. The Schlenk tube wasevacuated and flushed with thy nitrogen three times. Deoxygenatedtoluene (28 mL) was added to the Schlenk tube. The resulting solutionwas deoxygenated via three freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.05 g, 0.38 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 50° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis. Conversion was followed by ¹H NMR spectrometry and molecularweight analysis by SEC.

The polymer was purified by the dropwise addition of the reactionsolution to a vigorously stirred solution of diethyl ether (400 mL). Theresulting white powder was filtered, dissolved in toluene (20 mL) andprecipitated in diethyl ether (400 mL). This procedure was repeatedthree times.

TABLE 2 Data for the polymerization of methoxypolyethyleneglycolmethacrylate (2080) with an initiator derived from N-hydroxy succinimideat 50° C. in 80% toluene solution. Sample Time/minutes Conversion^(a)/%Mn^(b) PDi^(b) 86 7 8700 1.06 289 12 10920 1.07 899 24 14450 1.05 136733 15810 1.04 2758 45 20220 1.07 3962 53 23180 1.07 ^(a)Conversion wasdetermined using 1H NMR. ^(b)Molecular mass determined by SEC using PMMAstandards.

Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycolmethacrylate) was freeze dried prior to use to remove all water.

[PEG]/[I]/[Cu]/[L]=19.2/1/1/2 in 80% Toluene Solution (AJ U2-27c)@90° C.

N-hydroxy succinimide initiator, (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g,0.189 mmol, 1 eq) and methoxypolyethyleneglycol methactylate (PEG)(average molecular weight=2080, 7.55 g, 3.63 mmol), and a magneticfollower were placed in an oven dried Schlenk tube. The Schlenk tube wasevacuated and flushed with dry nitrogen three times. Deoxygenatedtoluene (2 mL) was added to the Schlenk tube. The resulting solution wasdeoxygenated via three freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.05 g, 0.38 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 90° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis. Conversion was followed by ¹H NMR spectrometry and molecularweight analysis by SEC.

The polymer was purified by the dropwise addition of the reactionsolution to a vigorously stirred solution of diethyl ether (400 mL). Theresulting white powder was filtered, dissolved in toluene (20 mL) andprecipitated in diethyl ether (400 mL). This procedure was repeatedthree times.

TABLE 3 Data for the polymerization of methoxypolyethyleneglycolmethacrylate (2080) with an initiator derived from N-hydroxy succinimideat 90° C. in 80% toluene solution. Sample Time/minutes Conversion^(a)/%Mn^(b) PDi^(b) 86 18 11100 1.08 289 26 14870 1.08 899 31 17900 1.08 136735 18110 1.09 2758 38 18110 1.09 3962 39 18240 1.08 ^(a)Conversion wasdetermined using 1H NMR. ^(b)Molecular mass determined by SEC using PMMAstandards.

Bisomer S20W (50% aqueous solution of methoxypolyethyleneglyeolmethacrylate) was freeze dried prior to use to remove all water.

[PEG]/[I]/[Cu]/[L]=23.9/1/1/2 in 66% Toluene Solution (AJ U2-11) @90° C.

N-hydroxy succinimide initiator, (23 g, 9.47 mmol), Cu(I)Br (1.35 g,9.47 mmol, 1 eq) and methoxypoiyethyleneglycol methacrylate (PEG)(average molecular weight=628, 142.0 g, 0.226 mol), and a magneticfollower were placed in an oven dried Schlenk tube. The Schlenk tube wasevacuated and flushed with dry nitrogen three times. Deoxygenatedtoluene (261 mL) was added to the Schlenk tube. The resulting solutionwas deoxygenated via three freeze pump thaw cycles and then degassedN-propyl-2-pyridylmethanimine: (2.80 g, 0.019 mol) was added. Thereaction was placed in a thermostatically controlled oil bath at 90° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis. Conversion was followed by ¹H NMR spectrometry andmolecular weight analysis by SEC.

The polymer was purified by the dropwise addition of the reactionsolution to a vigorously stirred solution of diethyl ether (1000 mL).The resulting oil was washed with diethyl ether (3×1000 mL) and thendried in vacuo.

TABLE 4 Data for the polymerization of methoxypolyethyleneglycolmethacrylate (628) with an initiator derived from N-hydroxy succinimideat 90° C. in 66% toluene solution. Sample Time/minutes Conversion^(a)/%Mn^(b) PDi^(b) 48 21 4449 1.11 132 40 7198 1.08 185 44 7779 1.07 245 468105 1.09 300 48 8331 1.09 ^(a)Conversion was determined using 1H NMR.^(b)Molecular mass determined by SEC using PMMA standards.

Bisomer MPEG550MA was used as provided.

Polymerisation of Methoxypolyethyleneglycol Methacrylate (1080) Usingthe N-Hydroxy Succinimide Derived Initiator[PEG]/[I]/[Cu]/[L]=13.9/1/1/2 in 66% Toluene Solution (AJ U2-13)@90° C.

N-hydroxy succinimide initiator, (0.526 g, 1.99 mmol), Cu(I)Br (0.29 g,2.02 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG)(average molecular weight=1080, 29.62 g, 0.027 mol), and a magneticfollower were placed in an oven dried Schlenk tube. The Schlenk tube wasevacuated and flushed with dry nitrogen three times. Deoxygenatedtoluene (60 mL) was added to the Schlenk tube. The resulting solutionwas deoxygenated via three freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.51 g, 3.96 mol) was added. The reactionwas placed in a thermostatically controlled oil bath at 90° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis. Conversion was followed by ¹H NMR spectrometry and molecularweight analysis by SEC.

The polymer was purified by the dropwise addition of the reactionsolution to a vigorously stirred solution of diethyl ether (1000 mL).The resulting oil was washed with diethyl ether (3×1000 mL) and thendried in vacuo.

TABLE 5 Data for the polymerization of methoxypolyethyleneglycolmethacrylate (1080) with an initiator derived from N-hydroxy succinimideat 90° C. in 66% toluene solution. Sample Conversion^(a)/ Time/minutes %Mn^(b) PDi^(b) 1250 47.3 12180 1.16 2460 50.4 12460 1.16 3890 52.8 125401.20 ^(a)Conversion was determined using 1H NMR. ^(b)Molecular massdetermined by SEC using PMMA standards.

[PEG]/[I]/[Cu]/[L]=9.3/1/1/2 in 66% Toluene Solution (AJ U2-15) @90° C.

N-hydroxy succinimide initiator, (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g,1.89 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG)(average molecular weight=1080, 18.90 g, 0.018 mol), and a magneticfollower were placed in an oven dried Schlenk tube. The Schlenk tube wasevacuated and flushed with dry nitrogen three times. Deoxygenatedtoluene (35 mL) was added to the Schlenk tube. The resulting solutionwas deoxygenated via three freeze pump thaw cycles and then degassedethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The reactionwas placed in a thermostatically controlled of bath at 90° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis. Conversion was followed by ¹H NMR spectrometry and molecularweight analysis by SEC.

TABLE 6 Data for the polymerization of methoxypolyethyleneglycolmethacrylate (1080) with an initiator derived from N-hydroxy succinimideat 90° C. in 66% toluene solution. Sample Conversion^(a)/ Time/minutes %Mn^(b) PDi^(b) 4160 88.7 9870 1.22 ^(a)Conversion was dertimined using1H NMR. ^(b)Molecular mass determined by SEC using PMMA standards.

Bisomer S10W (50% aqueous solution of methoxypolyethyleneglycolmethacrylate) was freeze dried prior to use to remove all water.

Polymerisation of Methxrypolyethyleneglycol Methacrylate (628) Using theHydroxy Succinimide Derived Initiator [PEG]/[I]/[Cu]/[L]=6.4/1/1/2 in66% Toluene Solution (AJ U2-31a) @30° C.

N-hydroxy succinimide initiator, (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g,1.89 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG)(average molecular weight=628, 7.57 g, 0.012 mol), and a magneticfollower were placed in art oven dried Schlenk tube. The Schlenk tubewas evacuated and flushed with dry nitrogen three times. Deoxygenatedtoluene (14 mL) was added to the Schlenk tube. The resulting solutionwas deoxygenated via three freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 30° C. (r=0) andsamples were removed periodically for conversion and molecular weightanalysis. Conversion was followed by ¹H NMR spectrometry and molecularweight analysis by SEC.

TABLE 7 Data for the polymerization of methoxypolyethyleneglycolmethacrylate (628) with an initiator derived from N-hydroxy succinimideat 30° C. in 66% toluene solution. Sample Conversion^(a)/ Time/minutes %Mn^(b) PDi^(b) 60 19 2850 1.04 131 32 3230 1.10 199 45 3560 1.12 250 533760 1.12 298 56 3980 1.12 ^(a)Conversion was determined using 1H NMR.^(b)Molecular mass determined by SEC using PMMA standards.Bisomer MPEG550MA was used as provided.[PEG]/[I]/[Cu]/[L]=6.4/1/1/2 in 66% toluene solution (AJ U2-31b) @50° C.

N-hydroxy succinimide initiator, (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g,1.89 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG)(average molecular weight=628, 7.57 g, 0.012 mol), and a magneticfollower were placed in an oven dried Schlenk tube. The Schlenk tube wasevacuated and flushed with dry nitrogen three times. Deoxygenatedtoluene (14 mL) was added to the Schlenk tube. The resulting solutionwas deoxygenated via three freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 50° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis. Conversion was followed by ¹H NMR spectrometry and molecularweight analysis by SEC.

TABLE 8 Data for the polymerization of methoxypolyethyleneglycolmethacrylate (628) with an initiator derived from N-hydroxy succinimideat 50° C. in 66% toluene solution. Sample Conversion^(a)/ Time/minutes %Mn^(b) PDi^(b) 59 39 3212 1.09 126 56 3958 1.11 195 69 4375 1.13 246 754649 1.13 295 82 4874 1.13 ^(a)Conversion was determined using 1H NMR.^(b)Molecular mass determined by SEC using PMMA standards.

Bisomer MPEG550MA was used as provided.

EXPERIMENTAL General Experimental

For all following polymerisations conversion data was obtained by ¹H NMRspectroscopy and molecular weight data (Mn and PDi) by SEC using PMMAstandards.

Methoxypolyethyleneglycol methacrylates were obtained from Sigma-Aldrichor Laporte Performance Chemicals and used either as received(MPEG(395)MA: Mn 475 g mol⁻¹ and BISOMER MPEG(550)MA: Mn 628 g mol⁻¹) orfreeze dried prior to use to remove all water (BISOMER S10WMPEG(1000)MA: Mn 2080 g mol⁻¹ and BISOMER S20W MPEG(2000)MA: Mn=2080 gmol⁻¹).

The ligands N-(n-alkyl)-2-pyridylmethanimine were prepared as describedpreviously.¹ Copper(I) bromide was purified as necessary by a methodbased on that of Keller and Wycoff.²

All other reagents were obtained from either Sigma-Aldrich, Romil,Fisher or Acros and used as received.

Functional Initiators

The following table lists the functional initiators used to polymerisethe methoxypolyethyleneglycol methacrylates.

TABLE 1 Functional initiators. Initiator code Initiator structure  8

 7

 5

 9

 6

10

11

12

13

14

15

Functional Polymers

The following table lists the functional polymers prepared usingmethoxypolyethyleneglycol methacrylates and the initiators shown inTable 1. These polymers can be used either directly to react with usefulbiomolecules or converted simply into new macromolecules that will reactwith useful biomolecules.

TABLE 2 Functional polymers. Initiator used Polymer structure  8

 7

 5

 9

 6

10

11

12

13

14

15

Preparation of Initiators and Intermediates Preparation of Initiator 8N-hydroxysuccinimde-2-bromopropionate

N-hydroxysuccinimide (4.51 g, 39.22 mmol) and 2-bromopropionic acid (2.9mL, 32.68 mmol) were dissolved in anhydrous DCM (1000 ml) in a 2000 mLround-bottomed flask under nitrogen equipped with a magnetic stirrer.The flask was then cooled to 0° C. with an ice bath before the dropwiseaddition of a solution of N,N′-Dicyclohexylcarbodiimide (6.70 g, 32.68mmol) in 50 mL of anhydrous DCM. After addition, the mixture was stirredat room temperature overnight. The reaction mixture was then filteredand the solvent evaporated to give a yellow solid that was purified byflash chromatography (CC, SiO₂, Et₂OO, R_(f(ester))=0.31). Obtained 7.2g (28.91 mmol, 74%) of product as a white solid. Melting point: 69-70°C. ¹H NMR (CDCl₃) δ (ppm) 1.96 (d, 3H, CH(CH)Br, J=6.78 Hz), 2.86 (s,4H, H_(cycl)), 4.61 (q, 1H, CH(CH₃)Br, J=7.03 Hz). ¹³C NMR (CDCl₃) δ(ppm) 21.67 (1C, CH(CH ₃)Br) 25.74 (2C, C_(cycl)), 34.97 (1C,CH(CH₃)Br), 166.17 (1C, C═O), 168.69 (2C, C_(cycl)═O). IR (solid, ATRcell) ν (cm⁻¹) 1808, 1781 (C_(cycl)═O), 1729 (C═O). Mass spectroscopy(+EI, m/z) 248.964. Elem. Anal. Theoretical for C₇H₈NO₄Br: C, 33.62; H,3.22; N, 5.60. Found: C, 33.47; H, 3.16; N, 5.46.

Preparation of Initiator 7N-hydroxysuccinimide-2-bromo-2-methylpropionate

N-Hydroxysuccinimide (11.51 g, 0.1 mol) was dissolved in anhydrousdichloromethane (100 ml) with triethylamine (28.1 mL, 0.2 mol) undernitrogen in a 250 ml round-bottomed flask equipped with a magneticstirrer. The flask was cooled to 0° C. with an ice bath before thedropwise addition of 2-bromo-2-methylpropionyl bromide (13.9 mL, 0.11mol). Next the mixture was stirred for 45 minutes and allowed to reachroom temperature. After this the reaction mixture was poured into anexcess of cold water and extracted with diethyl ether (3×50 mL). Theorganic layer was subsequently washed with a saturated aqueous solutionof sodium carbonate (3×50 mL), acidified water (pH=4.5, 3×50 mLl), andagain the saturated aqueous solution of sodium carbonate (3×50 mL). Theorganic layer was dried over anhydrous magnesium sulphate and filtered.Finally the solvent was removed under reduced pressure by using therotary evaporator in order to isolate the title compound in quantitativeyield as a white solid. ¹H NMR (CDCl₃) δ (ppm) 2.08 (s, 6H, C(CH ₃)₂Br),2.87 (s, 4H, H_(cycl)). ¹³C NMR (CDCl₃) δ (ppm) 26.03 (2C, C_(cycl)),31.09 (2C, C(CH₃)₂Br), 51.60 (1C, (CH₃)₂Br), 167.89 (1C, C═O), 169.02(2C, C_(cycl)═O). IR (solid, ATR cell) ν (cm⁻¹) 1803, 1772 (C_(cycl)═O),1728 (C═O), 1394, 1359, 1197, 1121, 1071, 996, 924, 856, 811, 731, 648.Mass spectroscopy (+EI, m/z) 266, 265, 156, 151, 149, 123, 121, 116,115, 91, 87, 70, 69. Elem. Anal. Theoretical for C₈H₁₀NO₄Br: C, 36.39;H, 3.82; N, 5.30; Br, 30.26. Found: C, 36.35; H, 3.82; N, 5.03; Br,30.17. Melting point 72-74° C.

Preparation of Initiator 5 2,4-Dichloro-6-methoxy-1,3,5-triazine³

To 200 ml of methanol and 25 ml of water were added 33.6 g. (0.4 mole)of sodium bicarbonate and 36.8 g. (0.2 mole) of cyanuric chloride. Thismixture was stirred at 30° C. for 30 minutes until the evolution ofcarbon dioxide had nearly ceased, and water was then added. Thecrystalline solid which separated was filtered, washed with water, anddried in a vacuum desiccator. The yield of crude2,4-dichloro-6-methoxy-triazine was 10.5 g. (58%), m.p. 87-89° C. Afterrecrystallization from heptane the m.p. was 88-90° C. Elem. Anal. Calcd.for C₄H₃N₃OCl₂: C, 26.67; H, 1.67; N, 23.35; Cl, 39.44. Found: C, 26.96;H, 1.84; N, 23.25; Cl, 39.19.

4-[(4-chloro-6-methoxy-1,3,5-trazine-2-yl)amino]phenol

A solution of 2,4-dichloro-6-methoxy-1,3,5-triazine (9.00 g, 50.0 mmol)in 100 mL of acetone was cooled to 0° C. and, under stirring, solid4-amino phenol (5.46 g, 50.0 mmol) was added in small portion, over ca 2min. The white suspension was then let to warm to room temperature andstirred for further 1 h, while being neutralized with a 2 M aqueoussolution of Na₂CO₃ during the reaction. The mixture was then poured into500 mL of ice/water and the resulting white precipitate was filtered anddried, to give 9.6 g (38.0 mmol, yield 76%) of4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenol that can be usedwithout further purifications. An analytical sample can be obtained byflash chromatography (CC, SiO₂, petroleum ether/Et₂O 1:1, Rf=: 0.14).The NMR analysis (DMSO d6) reveals the presence, in solution, of 2rotational isomers (molar ratio 7:3). M.p was 172° C. IR ν_((NH)) 3476cm⁻¹. ν_((OH)) 3269 cm⁻¹. Major isomer: ¹H NMR (DMSO d6) δ=3.94 (s, 3H,OCH₃); 6.79 (d, J=8.8 Hz, 2H, CH Ar), 7.48 (d, J=8.8 Hz, 2H, CH Ar),9.40 (s, 1H, OH), 10.46 (s, 1H, NH). ¹³C NMR (DMSO d6) δ 55.52 (1C,OCH₃); 115.46 (2C, CH Ar), 123.91 (2C, CH Ar), 129.49 (1C, C Ar), 154.44(1C, C Ar), 164.81 (1C, C Ar), 169.57 (1C, C Ar), 171.23 (1C, C Ar).Minor isomer: ¹H NMR (DMSO d6) δ 3.96 (s, 3H, OCH₃); 6.79 (d, J=8.9 Hz,2H, CH Ar), 7.39 (d, J=8.9 Hz, 2H, CH Ar), 9.42 (bs, 1H, OH), 10.10.32(s, 1H, NH). ¹³C NMR (DMSO d6) δ=55.10 (1C, OCH₃); 115.46 (2C, CH Ar),123.03 (2C, CH Ar), 129.26 (1C, C Ar), 154.76 (1C, C Ar), 165.20 (1C, CAr), 170.48 (1C, C Ar), 170.64 (1C, C Ar).

4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenyl-2-bromo-2-methylpropionate

A solution of 2-bromoisobutryl bromide (1.0 mL, 7.90 mmol) in 20 mL ofTHF was added dropwise to a solution of4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenol BIW009 (1.9 g,7.52 mmol) and triethylamine (1.1 mL, 8.0 mmol) in 100 mL of THF, at−10° C. During the dropping (ca. 15 min) the precipitation oftriethylammonium bromide was observed. The reaction was monitored by TLC(SiO₂, petroleum ether/Et₂O 1:1, BIW009 (starting material) Rf=0.14;BIW010 (final product) Rf=0.26). After 1.5 h the white suspension waspoured into a conical flask containing 150 mL of Et₂O and the ammoniumsalt removed by filtration on a sintered glass frit. The solvent wasthen evaporated at reduced pressure to give a white crude residue thatwas suspended in 10 ml of pentane and filtered. Obtained 2.56 g (6.37mmol, yield 85%) of BIW010 as white solid. The NMR analysis (DMSO d6)revealed the presence, in solution, of 2 rotational isomers (molar ratio7:3). M.p. 107-108° C. IR ν_((NH)) 3365 cm⁻¹. ν_((C|O)) 1747 cm⁻¹. Majorisomer: ¹H NMR (DMSO d6) δ 2.05 (s, 6H, C(CH₃)₂Br), 3.96 (s, 3H, OCH₃),7.17 (d, J=8.9 Hz, 2H, CH Ar), 7.77 (d, J=8.9 Hz, 2H, CH Ar), 10.78 (s,1H, NH). ¹³C{¹H} NMR (DMSO d6) δ=30.42 (2C, CH₃), 55.75 (bs, 1C, OCH₃),57.29 (1C, C(CH₃)₂Br), 121.96 (2C, CH Ar), 122.12 (2C, CH Ar), 136.29(1C, C Ar), 146.61 (bs, 1C, C Ar), 165.10 (bs, 1C, C Ar), 169.89 (bs,1C, C Ar), 170.16 (1C, OC(O)C(CH₃)₂Br), 171.33 (bs, 1C, C Ar). Minorisomer. ¹H NMR (DMSO d6) δ=2.05 (s, 6H, C(CH₃)₂Br), 3.96 (s, 3H, OCH₃),7.17 (d, J=8.9 Hz, 2H, CH Ar), 7.69 (d, J=8.9 Hz, 2H, CH Ar), 10.66 (s,1H, NH).

¹³C NMR (DMSO d6) δ=30.42 (2C, CH₃), 55.75 (bs, 1C, OCH₃), 57.29 (1C,C(CH₃)₂Br), 121.96 (2C, CH Ar), 122.73 (2C, CH Ar), 136.29 (1C, C Ar),146.61 (bs, 1C, C Ar), 165.10 (bs, 1C, C Ar), 169.89 (bs, 1C, C Ar),170.16 (1C, OC(O)C(CH₃)₂B3r), 171.33 (bs, 1C, C Ar).

Preparation of Initiator 9 2-Hydroxyethyl-2-bromo-2-methylpropionate

Ethylene glycol (279 g, 4500 mmol) and Et₃N (3.34 g, 33.0 mmol) werepoured in a 2-necked round bottom flask. To this was added dropwise anda solution of 2-bromoisobutryl bromide (6.90 g, 30.0 mmol) in anhydrousTHF (50 mL) at room temperature over ca. 1 h. The colourless solutionwas stirred overnight, then diluted in 500 mL of water and extractedwith 3×200 mL of a mixture of Et₂O/CH₂Cl₂ (4:1). The organic layers,reunited, were washed with 2×200 mL of water and dried over MgSO₄.Evaporation of the solvent at reduced pressure (rotavapor, withoutheating) gave a pale yellow liquid. The latter was dissolved in ca. 30mL of CH₂Cl₂ then 10 g of SiO₂ were added and the solvent evaporatedagain until a white powder was obtained. This was poured in a columnpacked with SiO₂, (ca 15 cm depth) previously eluted with petroleumether/Et₂O 5:1 and purified by column chromatography (elute withpetroleum ether/Et₂O 5:1) to eliminate the impurities. The desiredproduct, using this solvent mixture, has an Rf˜0 (stays on the bottom ofthe TLC plate). When the impurities have been eliminated, the column waseluted with 100% Et₂O, to give a colourless liquid. Yield 82%. IRν_((N—H)) 3388 cm⁻ (broad); ν_((C═O)) 1731 cm⁻¹. ¹H NMR (CDCl₃) δ=1.97(s, 6H, CH₃); 3.89 (t, J=4.6 Hz; 2H, OCH₂CH ₂OH); 4.33 (t, J=4.6 Hz; 2H,OCH ₂CH₂OH). ¹³C NMR (CDCl₃) δ=30.45 (2C, CH₃), 55.55 (1C, C(CH₃)₂Br);60.66 (1C, OCH₂ CH₂OH); 65.90 (1C, OCH₂CH₂OH); 171.69 (1C, C═O).

Preparation of Initiator 6 2-Phthalmidoethyl-2-bromo-2-methylpropionate

N-(2-hydroxyethyl)phthalimide (19.12 g, 0.1 mol) was dissolved inanhydrous THF (250 mL) with triethylamine (28.1 mL, 0.2 mol) undernitrogen in a 500 mL round bottom flask equipped with a magnetic stirrerand dropping funnel. The flask was cooled to 0° C. with an ice/salt bathbefore the dropwise addition of 2-bromo-2-methylpropionyl bromide (13.9mL, 0.11 mol). The mixture was stirred for 45 minutes and allowed toreach room temperature before the mixture was poured into an excess ofcold water and the product extracted with diethyl ether (3×100 mL). Theorganic layer was subsequently washed with a saturated aqueous solutionof sodium carbonate (3×100 mL), acidified water (pH 4.6, 3×100 mL) andagain with saturated aqueous solution of sodium carbonate (3×100 mL).The organic layer was dried over anhydrous magnesium sulphate andfiltered. The product was isolated via reduction under reduced pressureto obtain a white solid (25.79 g, 75.8% yield). ¹H NMR (CDCl₃) δ (ppm)1.81 (s, 6H, C(Ct)₂Br), 3.95 (t, 2H, J=5.3 Hz, CH ₂—N), 4.35 (t, 2H,J=5.4 Hz, CH ₂—O), 7.67 (m, 2H, H_(aro)), 7.78 (m, 2H, H_(aro)). ¹³C NMR(CDCl₃) δ (ppm) 31.00 (2C, C(CH₃)₂Br), 37.12 (1C, CH₂—N), 55.92 (1C,C(CH₃)₂Br), 63.42 (1C, CH₂—O), 123.78 (2C, C_(aro)), 132.35 (1C, C^(IV)_(aro)), 133.54 (2C, C_(aro)), 168.40 (2C, C_(cycl)═O), 171.87 (1C,C═O). IR (solid, ATR cell) ν (cm⁻¹) 2975, 1774 (C_(cycl)═O), 1705 (C═O),1417, 1392, 1321, 1276, 1158, 1105, 1063, 985, 763, 716, 632. Meltingpoint 63-65° C.

Preparation of Initiator 10 Tritylthiolether Propanol

Sodium hydride (10.95 g, 0.273 mol, 60% in oil) was suspended in THF(750 mL) at 0° C. Triphenylmethanethiol (75.5 g, 0.273 mol) in THF (600ml) was added to the suspension and stirred at 0° C. for 10 minutes.3-Bromo-1-propanol (24.75 mL, 0.273 mol) in THF (300 mLl) was added andthe mixture stirred at 0° C. for 20 minutes. After this time TLC showedmostly one major product (Rf approx 0.3 ethyl acetate/hexane 1:9). Waterwas added and the product extracted into ethyl acetate (2×1000 mL) (anaqueous NaCl solution was used to break up the emulsion), dried withsodium sulfate, filtered and concentrated. The product was purified bycolumn chromatography using ethyl acetate/hexane 1:9 to 1:4 as theeluent (SiO₂) to give a white solid, which was triturated with hexaneand filtered to give 65.2 g of product. ¹H NMR (CDCl₃) δ ppm 7.6-7.1 (m,15H H_(aro)), 3.58 (t, 2H, CH ₂OH), 2.29 (t, 2H, SCH₂), 1.65 (q, 2H,CH₂CH ₂CH₂), 1.45 (OH).

3-Tritylthioletherpropyl-2-bromo-2-methylpropionate

Tritylthiol ether propanol (50 g, 0.150 mol), triethylamine (31.3 mL,0.225 mol) and anhydrous tetrahydrofuran (125 mL) were placed in athree-necked round bottom flask containing a magnetic follower andfitted with a pressure equalizing dropping funnel. The flask was cooledwith the use of an ice bath and 2-bromoisobutyrl bromide (27.8 mL, 0.225mol) was added to the dropping funnel. Whilst stirring the2-bromoisobutyrl bromide was added drop-wise to the cooled solution andthe solution left stirring over night. The mixture is then filtered toremove the triethylamine hydrochloride salt before the addition ofdichloromethane (500 mL) and subsequently washed with dilutehydrochloric acid (2×300 mL), dilute sodium hydroxide (2×300 mL) andfinally distilled water (3×300 mL). The Organic layer was separated andthe product isolated by flash evaporation of solvent, the product wasthen triturated with hexane, filtered and the product collected inquantitative yield. ¹H NMR (CDCl₃) δ ppm 7.4-7.1 (m, 15H, H_(aro)), 4.02(t, 2H, CH₂CO₂), 2.15 (t, 2H, SCH₂), 1.78 (s, 3H, C(CH₃)₂Br), 1.63 (q,2H, CH₂CH ₂CH₂).

Preparation of Initiator 11 4-(2-bromo-2-methylpropionate) benzaldehyde

4-Hydroxybenzaldehyde 12.21 g (0.1 moles), triethylamine, 15.3 mL (0.11moles) and anhydrous THF 400 mL were placed in a 3 neck round bottomedflask, Bromoisobutyryl bromide 13.6 mL (0.11 moles) was added slowlywith stirring. A white precipitate of triethylammonium bromide wasformed. The mixture was left to react for 6 hours with stirring. Oncompletion of the reaction triethylammonium bromide was removed byfiltration and the THF was removed by rotary evaporation. The resultingorange liquid was dissolved in dichloromethane and subsequently washedwith 2×200 mL portions of saturated Na₂CO₃ (aq), dil. HCl (aq) anddistilled water. The dichloromethane was dried using MgSO₄ and thesolvent removed by rotary evaporation to give a yellow oily liquid whichcrystallised upon standing. This was recrystalised from diethyl ether×2.Yield=18.95 g (69.9%). ¹H NMR (CDCl₃) δ (ppm) 10.00 (s, 1H, CHO), 7.94(d, J=4.6 Hz, 2H, H_(aro)), 7.31 (d, J=4.8 Hz 2H, H_(aro)), 2.06 (s, 6H,C(CH ₂Br). ¹³C NMR (CDCl₃) δ (ppm) 190.59, 169.33, 155.08, 134.07,131.02, 121.71, 54.94, 30.25. IR (Solid, ATR Cell) 2984, 2820, 2730(O═C—H), 1746 (C═O), 1693 (H—C—O), 1590, 1500, 1374, 1262, 1207, 1153,1132, 1099, 1009, 932, 881, 808, 658: +EI MS (m/z) 273, 271 (masspeaks), 210, 193, 163, 151, 149, 140, 123, 121, 102. Elem. Anal.Theoretical for H11O3Br: C=48.73, H=4.09. Found: C=48.63, H=4.03.

Preparation of Initiator 12 2-(2,2-Dimethoxy-ethoxy)-ethanol

Potassium hydroxide (30 g, 0.51 mol) was suspended in ethylene glycol(100 ml) and the mixture was heated to 115° C. with stirring. After theKOH dissolved completely, 2-chloro-1,1-dimethoxy-ethane (30.0 mL, 0.263mol) was added dropwise (ca. 30 minutes) and the solution was stirred at115° C. for 72 h. The resulting suspension was cooled to roomtemperature and 150 mL water was added. The solution was extracted withdichloromethane (3×100 mL) and the organic layers combined was washedwith brine (2×100 mL) and dried with MgSO₄. After filtration the solventwas removed under reduced pressure to give the product as a yellow oil(Yield, 17.7 g, 44.9%). ¹H NMR (400.03 MHz, CDCl₃, 298 K) δ=2.20 (s, 1H,OH), 3.40 (s, 6H, OCH ₃), 3.55 (d, J=5.3 Hz, 2H, CHCH ₂), 3.63-3.61 (m,J=4.0 Hz, OCH ₂), 3.74-3.72 (m, J=4.0 Hz, 2H, CH ₂OH), 4.52 (t, 1H,CH(OCH₃)₂. ¹³C{H} NMR (100.59 MHz, CDCl₃, 298 K) δ=54.12 (2C, CH₃),61.82 (1C, CH₂OH), 70.78 (1C, CHCH₂O), 73.00 (1C, OCH₂CH₂), 102.73 (1C,CH). Anal. Calcd. for C₆H₁₄O₄: C, 47.99; H, 9.40. Found C, 45.02; H,8.74

2-Bromo-2-methyl-propionic acid 2-(2,2-dimethoxy-ethoxy)-ethyl ester

A solution of 2-(2,2-dimethoxy-ethoxy)-ethanol (11 g, 0.073 mol) andtriethylamine (12 mL, 0.088 mol) in dichloromethane (150 mL) was cooledto 0° C. and a solution of 2-bromo-2-methyl propionyl bromide (8.5 mL,0.069 mol) in 50 mL of dichloromethane was added dropwise in ca. 30 min.After stirring overnight at room temperature the resulting suspensionwas filtered and the yellow solution was washed with saturated NaHCO₃aqueous solution (2×100 mL) and dried with MgSO₄. After filtration thesolvent was removed under reduced pressure and the yellow oily residuewas purified by distillation (b.p. 70° C./0.02 mbar) to give 14.0 g(0.061 mol, yield: 89%) of product as a colourless oil. ¹H NMR (400.03MHz, CDCl₃, 298 K) δ=1.94 (s, 6H, (CH ₃)₂CBr), 3.39 (s, 6H, OCH₃), 3.56(d, J=5.3 Hz, 2H, CHCH ₂), 3.76 (t, J=4.8 Hz, CH₂OCH ₂), 4.33 (t, J=4.8Hz, 2H, CH ₂OCO), 4.50 (t, 1H, CH(OCH₃)₂. ¹³C{¹H} NMR (100.59 MHz,CDCl₃, 298 K) δ=30.90 (2C, C(CH₃)₂), 54.13 (2C, CH₃O), 55.79 (1C,BrC(CH₃)₂), 65.24 (1C, CH₂OC(═O)), 69.21 (1C, CHCH₂O), 71.18 (1C,OCH₂CH₂), 102.83 (1C, CH).

Preparation of Initiator 133-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid

A solution of maleic anhydride (5.00 g, 0.0561 mol) in acetic acid (70ml) was added dropwise to a solution of β-alanine (5.50 g, 0.0561 mol)in acetic acid (25 ml) and the mixture was stirred at room temperaturefor three hours. 50 mL of acetic acid was added to the white suspensionand the mixture heated up to 115° C. After 1 h a limpid colourlesssolution was observed. The mixture was then stirred at this temperatureovernight and the colour turned to orange. The solvent was then removedunder reduced pressure and 30 mL of toluene was then added to theresulting orange oil. This was then evaporated under reduced pressureand this operation was repeated 3 times. The orange residue was thenpurified by flash chromatography (CC, SiO₂, CH₂Cl₂/ethyl acetate 9:1) togive the product as a white solid (3.86 g, 0.0228 mol, 41%). m.p.105-107° C. IR (neat): 3092, 2883, 2537, 1695, 1445, 1411, 1373, 1337,1305, 1230, 1151, 1081, 1043, 924, 830, 773, 694, 618 cm⁻¹. ¹H NMR(400.03 MHz, CDCl₃, 298 K) δ=2.69 (t, J=7.3 Hz, 211, CH₂), 3.82 (t,J=7.3 Hz, 2H, CH₂), 6.71 (s, 2H, CH_(vinyl)), 10.07 (bs, 1H, COOH).¹³C{¹H} NMR (100.59 MHz, CDCl₃, 298 K) δ=32.62 (1C, CH₂), 33.36 (1C,CH₂), 134.38 (2C, CH_(vinyl)), 170.48 (1C, C), 176.64 (2C, C). Anal.Calcd. for C₇H₇NO₄: C, 49.71; H, 4.17; N, 8.28. Found C, 49.35; H, 4.19;N, 7.95.

3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-proponyl chloride(3-malemidopropionyl chloride)

3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid (2.20 g, 0.0130mol) was dissolved in CH₂Cl₂ (100 mL). Oxalyl chloride (1.1 mL, 0.0130mol) was then added, at room temperature. 50 μL of DMF were addeddropwise and an intense evolution of gas was observed. The solution wascolourless before the addition of DMF and remained colourless for 1 h,then slowly turned to very pale yellow (but still very limpid). After 3h the solvent was removed under reduced pressure to give an off-whitesolid that became pale brown after standing under vacuum at roomtemperature for 1 h. The acid chloride product so obtained was useddirectly without further purifications. IR (neat): 3095, 1803 1698,1446, 1410, 1387, 1360, 1307, 1230, 1148, 1131, 1083, 1011, 948, 922,833, 719, 689, cm⁻¹. ¹H NMR (400.03 MHz, CDCl₃, 298 K) δ=3.25 (t, J=6.9Hz, 2H, CH₂), 3.86 (t, J=6.9 Hz, 2H, CH₂), 6.73 (s, 2H, CH_(vinyl)).¹³C{¹H} NMR (100.59 MHz, CDCl₃, 298 K) δ=33.21 (1C, CH₂), 44.97 (1C,CH₂), 134.47 (2C, CH_(vinyl)), 170.08 (2C, C), 171.50 (1C, C).

2-Bromo-2-methyl-propionic acid2-[3-(2,5-dioxo-2,S-dihydro-pyrrol-1-yl)-propionyloxy]-ethyl ester

2-Hydroxyethyl-2-bromo-2-methylpropionate (initiator 9) (0.187 g, 0.887mmol) and 3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid (0.300 g,1.77 mmol) were dissolved in dichloromethane (10 ml) under nitrogen in a25 ml round-bottomed flask. Then N,N′-Dicyclohexylcarbodiimide (DCC)(0.366 g, 1.77 mmol) was added to the solution. After one day at roomtemperature, very low conversion was observed and 0.5 ml (9.50×10⁻³mmol) of a solution of 4-dimethylaminopyridine (DMAP) in dichloromethane(Conc._(DMAP)=19 mmol/l) was added. Total conversion was then achievedin 12 hours and the solvent was removed under reduced pressure. Thesolid residue was extracted with 3×50 ml of petroleum ether and thepetroleum ether was removed by evaporation under vacuum in order toisolate a colourless oil (0.270 g, 0.745 mmol, 84%). The pale pinkresidue was extracted with 3×50 ml of diethylether, but TLC(CH₂Cl₂/AcOEt9:1) revealed that only traces of the ester were present. An analyticalpure sample was obtained by flash chromatography (cc, SiOz, Pet.Ether/Et₂O 3:1).

¹H NMR (CDCl₃) δ (ppm) 1.88 (s, 6H, C(Ch)₂Br), 2.62 (t, 2H, CH₂—COO(CH₂)₂—O, J_(ab)=7.02 Hz), 3.78 (t, 2H, (CO)₂N—CH ₂, J_(ab)=7.07Hz), 4.27-4.35 (m, 4H, O—(CH ₂)₂—O), 6.68 (s, 2H, OC—CH═CH—CO). ¹³C NMR(CDCl₃) δ (ppm) 30.61 (2C, C(CH₃)₂Br), 32.75 (1C, CH₂—COO(CH₂)—O), 33.46(1C, (CO)₂N-CH₂), 55.45 (1C, C(CH₃)₂Br), 62.04 (1C, CH₂—COO—CH₂), 63.35(1C, CH₂—OOC—C(CH₃)₂Br), 134.25 (2C, OC—CH═CH—CO), 170.29 (1C, C═Oester), 170.40 (1C, C═O ester), 171.39 (2C, O═C—N(CH₂)—C═O). IR (solid,ATR cell) ν (cm⁻¹) 1769 ( ν _((C═O, imide))), 1736 ( ν _((C═O, acid))),1707 ( ν _((C═O, imide))).

Preparation of Initiator 14 2-Bromo-2-methyl-propionic acid3-tert-butoxycarbonylamino-propyl ester

A solution of 3-amino propanol (3.00 mL, 0.0392 mol) in 100 mL of THFwas cooled to 0° C. and Boc₂O (8.56 g, 0.0322 mol) in THF (50 mL) wasadded dropwise (ca. 20 min.). The solution was then warmed up to roomtemperature and stirred for 3 h. TLC analysis (SiO₂, 100% Et₂O) revealedthe complete disappearance of the amino alcohol starting material(R_(f)=0) and the presence of the expected N-Boc-protected amino alcoholintermediate (R_(f)=0.25). The mixture was then cooled to 0° C. and Et₃N(6.0 mL, 0.0431 mol) was added via syringe. A solution of 2-bromoisobutyryl bromide (4.85 mL, 0.0392 mol) in THF (50 mL) was addeddropwise in ca. 30 min. and the resulting white suspension stirred at 0°C. for 1 h and at room temperature for further 2 h. The mixture was thendiluted with Et₂O (200 mL) and the ammonium salt was filtered off andwashed with 3×50 mL of Et₂O. The colourless solution was washed with3×100 mL of water and dried over MgSO₄. Removal of the solvent underreduced pressure gave the product a colourless oil that was purified byflash chromatography (CC, SiO₂, Petroleum ether/Et₂O 8:1). Obtained10.42 g (0.0321 mol, 82%) of (1) as colourless oil. IR (neat): 3295,2976, 1734, 1713, 1695, 1517, 1463, 1391, 1366 1273, 1163, 1109, 1013,633 cm⁻¹. ¹H NMR (400.03 MHz, CDC₃, 298 K) δ=1.43 (s, 9H, CH₃), 1.88(quint., J=6.3 Hz, 2H, CH₂), 1.93 (s, 6H, CH₃), 3.23 (q, J=6.0 Hz, 2H,CH₂), 4.24 (q, J=6.0 Hz, 2H, CH₂), 4.77 (bs, 1H, NH). ¹³C {¹H} NMR(100.59 MHz, CDCl₃, 298 K) δ=28.54 (3C, CH₃), 28.99 (1C, CH₂), 30.88(2C, CH)), 37.57 (1C, CH₂), 55.95 (1C, C), 63.86 (1C, CH₂), 156.03 (1C,C), 171.89 (1C, C). Anal. Calcd. for C₁₂H₂₂BrNO₄: C, 44.46; H, 6.84; N,4.32; Br, 24.65. Found C, 44.48; H, 6.91; N, 4.33. Br, 24.91.

Preparation of Initiator 154,10-Dioxa-tricyclo[5.2.1.0^(2,6)]dec-8-ene-3,5-dione

Maleic anhydride (30.00 g, 0.306 mol) was suspended in 150 mL of tolueneand the mixture warmed to 80° C. Furan (33.4 mL, 0.459 mol) was addedvia syringe and the turbid solution stirred for 6 h. The mixture wasthen cooled to room temperature and the stirring was stopped. After 1 hthe resulting white crystals were filtered off and the solid washed with2×30 mL of petroleum ether. Obtained 44.40 g (0.267 mol, 87% yield) ofthe desired product as small white needless. m.p. 124-127° C. (dec.) IR(neat): 1857, 1780, 1309, 1282, 1211, 1145, 1083, 1019, 947, 920, 902,877, 847, 800, 732, 690, 674, 633, 575 cm⁻¹. ¹H NMR (400.03 MHz, CDCl₃,298 K) δ=3.17 (s, 2H, CH), 5.45 (t, J=1.0 Hz, 2H, CHO); 6.57 (t, J=1.0Hz, 2H, CH_(vinyl)). ¹³C{¹H} NMR (100.59 MHz, CDCl₃, 298 K) δ=48.85 (2C,CH), 82.35 (2H, CHO), 137.12 (2C, CH_(vinyl)), 170.04 (2C, CO). Anal.Calcd. for C₈H₆O₄: C, 57.84; H, 3.64. Found C, 57.74; H, 3.68.

4-(2-Hydroxy-ethyl)-10-oxa-4-aza-trcyclo[5.2.1.0^(2,6)]dec-8-ene-3,5-dione

The anhydride, 4,10-dioxa-tricyclo[5.2.1.0^(2,6)]dec-8-ene-3,5-dione,(2.00 g, 12.0×10⁻³ mol) was suspended in 50 mL of MeOH and the mixturecooled to 0° C. A solution of ethanolamine (0.72 mL, 12.0×10⁻³ mol) in20 mL of MeOH was added dropwise (10 min) and the resulting solution wasstirred for 5 min at 0° C., then 30 min at room temperature and finallyrefluxed for 4 h. After cooling to room temperature the solvent wasremoved under reduced pressure, the white residue was dissolved in 150mL of CH₂Cl₂ and washed with 3×100 mL of water. The organic layer wasdried over MgSO₄ and filtered. Removal of the solvent under reducedpressure furnished the desired product (1.04 g 5.0×10⁻³ mol, 42% yield)as white solid that was used for the next step without furtherpurifications. An analytical sample was obtained by flash chromatography(CC, SiO₂, 100% ethyl acetate, R_(f)(6)=0.26). m.p. 139-141° C. (dec).IR (neat): 3472, 1681, 1435, 1405, 1335, 1269, 1168, 1100, 1053, 1013,959, 916, 875, 850, 807, 722, 705, 654 cm⁻¹. ¹H NMR (400.03 MHz, CDCl₃,298 K) δ 1.90 (bs, 1H, OH); 2.90 (s, 2H, CH), 3.69-3.72 (m, 2H, CH₂),3.76-3.78 (m, 2H, CH₂), 5.28 (t, J=0.9 Hz, 2H, CH), 6.52 (t, J=0.9 Hz,2H, CH_(vinyl)). ¹³C{¹H} NMR (100.59 MHz, CDCl₃, 298 K) δ=41.77 (2C,NCH₂), 60.18 (2C, OCH₂), 47.50 (2C, CH), 81.04 (2C, CH), 136.60 (2C,CH_(vinyl)), 176.97 (2C, C). Anal. Calcd. for C₁₀H₁₁NO₄: C, 57.41; H,5.30; N, 6.70. Found C, 57.16; H, 5.37; N, 6.62.

2-Bromo-2-methyl-propionic acid2-(3,5-diaxo-10-oxa-4-aza-tricyclo[5.2.1.0^(2,6)]dec-8-en-4-yl)-ethylester

A solution of the alcohol,4-(2-hydroxy-ethyl)-10-oxa-4-aza-tricyclo[5.2.1.0^(2,6)]dec-8-ene-3,5-dione,(2.22 g, 10.6×10⁻³ mol) and Et₃N (1.6 mL, 11.7×10⁻³ mol) in 120 mL ofTHF (the solution remains slightly turbid) was cooled to 0° C. and asolution of 2-bromo isobutiryl bromide (1.4 mL, 11.1×10⁻³ mol) in 40 mLof THF was added dropwise (30 min). The white suspension was stirred for3 h at 0° C., then at room temperature overnight. The ammonium salt wasfiltered off and the solvent removed under reduced pressure to give apale-yellow residue that was purified by flash chromatography (CC, SiO₂,petroleum ether/ethyl acetate 1:1, R_(f)(7)=0.23). Obtained 3.54 g(9.88×10⁻³ mol, 93% yield) of initiator 15 as a white solid. m.p. 83-85°C. IR (neat): 1733, 1695, 1419, 1395, 1336, 1278, 1157, 1106, 1015, 874,852, 824, 724, 706, 654, 603 cm⁻¹. ¹H NMR (400.03 MHz, CDCl₃, 298 K)δ=1.86 (s, 6H, CH₃), 2.84 (s, 2H, CH), 3.78 (t, J=5.3 Hz, 2H, NCH₂);4.30 (t, J=5.3 Hz, 2H, OCH₂); 5.23 (t, J=1.0 Hz, 2H, CHO); 6.49 (t,J=1.0 Hz, 2H, CH_(vinyl)). ¹³C{¹H} NMR (100.59 MHz, CDCl₃, 298 K)δ=30.65 (2C, CH₂), 37.65 (2C, NCH₂), 47.56 (2C, CH), 55.80 (1C,C(CH₃)₂Br), 62.26 (OCH₂), 80.91 (2H, CHO), 136.62 (2C, CH_(vinyl)),171.46 (1C, CO_(ester)), 175.95 (2C, CO_(imide)). Anal. Calcd. forC₁₄H₁₆NO₅: C, 46.95; H, 4.50; N, 3.91; Br, 22.31. Found C, 46.88; H,4.55; N, 3.79; Br, 22.22

Preparation of PolyPEG Polymers Polymerisations Using Initiator 8

Polymerisation of MPEG(395)MA [PEG]/[I]/[Cu]/[L]=10/1/1/2 in 50v/v %Toluene Solution at 30° C.

A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27×10⁻³ mol),initiator 8 (0.569 g, 2.27×10⁻³ mol) and a magnetic follower prior tobeing deoxygenated by cycling between nitrogen and vacuum three times.To a second Schlenk tube was added MPEG(395)MA (10 mL, 22.74×10⁻³ mol),N-(n-ethyl)-2-pyridylmethanimine (0.64 mL, 4.54×10⁻³ mol) and toluene(10 mL). The mixture was immediately subjected to five freeze-pump-thawdegassing cycles. This solution was then transferred to the Schlenk tubecontaining the initiator and Cu(I)Br via a cannula. The resulting brownsolution was stirred at 30° C. Samples were removed periodically usingdegassed syringes and quenched in liquid nitrogen for conversion andmolecular weight analysis.

TABLE 3 Kinetic data for the polymerisation of MPEG(395)MA initiated by8 in toluene solution (50% v/v) at 30° C.([MPEG(395)MA]₀/[CuBr]₀/[NHSBr]₀/[ligand]₀ = 10/1/1/2). Con- Timeversion ln([M]₀/ M_(n,then) M_(n,SEC) Experiment (h) (%) [M]) (g ·mol⁻¹) (g · mol⁻¹) M_(w)/M_(n) Toluene 2 4.5 0.046 230 3040 1.07 Ethyl 417.3 0.190 870 3710 1.30 Ligand 6 30.2 0.359 1510 4480 1.13 8 40.2 0.5142010 4950 1.11 10 49.3 0.679 2470 5530 1.10 21 92.3 2.564 4620 7980 1.09

Polymerisation of MPEG(550)MA [PEG]/[I]/[Cu]/[L]=6.37/1/1/2 in 73 w/v %Toluene Solution at 50/70° C.

Initiator 8 (0.10 g, 0.400 mmol), Cu(I)Br (0.057 g, 0.400 mmol, 1 eq)and MPEG(550)MA, (1.60 g, 2.55 mmol), and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (5.90 mL)was added to the Schlenk tube. The resulting solution was deoxygenatedby bubbling with nitrogen for 1 hour and then degassedN-propyl-2-pyridylmethanimine (0.114 g, 0.797 mmol) was added. Thereaction was placed in a thermostatically controlled oil bath at 50° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis. The temperature was increased to 70° C. after 5 hours32 minutes.

TABLE 4 Data for the polymerization of MPEG(550)MA with initiator 8 at50/70° C. in 73 w/v % toluene solution. Sample Conversion/ Time/minutes% Mn PDi 385 15 4730 1.05 1357 42 10440 1.10 1663 45 10280 1.12

[PEG]/[I]/[Cu]/[L]=6.37/1/1/2 in 73 w/v % Toluene Solution at 90° C.

Initiator 8 (0.10 g, 0.400 mmol), Cu(I)Br (0.057 g, 0.400 mmol, 1 eq)and MPEG(550)MA (1.60 g, 2.341 mmol), and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (5.90 mL)was added to the Schlenk tube. The resulting solution was deoxygenatedby bubbling with nitrogen for 1 hour and then degassedN-propyl-2-pyridylmethanimine (0.114 g, 0.797 mmol) was added. Thereaction was placed in a thermostatically controlled oil bath at 90° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis.

TABLE 5 Data for the polymerization of MPEG(550)MA with initiator 8 at90° C. in 73 w/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 138 46 6950 1.09 240 46 7220 1.12 314 48 7370 1.12 1293 59 76901.14

[PEG]/[I]/[Cu]/[L]=31.9/1/1/2 in 67 v/v % Toluene Solution at 50° C.

Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) andMPEG(550)MA (8.0 g, 12.7 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (14.7 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.107 g, 0.80 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis. Thepolymer was purified by removing the solvent in vacuo and dialysisingthe residue using acidic water (pH ˜4). Subsequent freeze dryingisolated the product.

TABLE 6 Data for the polymerization of MPEG(550)MA with initiator 8 at50° C. in 67 v/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 120 7 7189 1.089 240 15 8976 1.074 360 20 10477 1.074 1320 8723051 1.147

[PEG]/[I]/[Cu]/[L]=6.4/1/1/2 in 67 v/v % Toluene Solution at 50° C.

Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) andMPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (3.0 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.107 g, 0.80 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 7 Data for the polymerization of MPEG(550)MA with initiator 8 at50° C. in 67 v/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 180 13 4984 1.064 360 31 6628 1.110 1320 86 11282 1.104

[PEG]/[I]/[Cu(I)]/[Cu(II)]/[L]=6.4/1/0.95/0.05/2 in 67 v/v % TolueneSolution at 50° C.

Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0545 g, 0.38 mmol, 0.95 eq),Cu(II)Br (0.0045 g, 0.02 mmol, 0.05 eq), and MPEG(550)MA (1.60 g, 2.55mmol), and a magnetic follower were placed in an oven dried Schlenktube. The Schlenk tube was evacuated and flushed with dry nitrogen threetimes. Deoxygenated toluene (3.0 mL) was added to the Schlenk tube. Theresulting solution was deoxygenated via three freeze pump thaw cyclesand then degassed N-ethyl-2-pyridylmethanimine (0.107 g, 0.80 mmol) wasadded. The reaction was placed in a thermostatically controlled oil bathat 50° C. (t=0) and samples were removed periodically for conversion andmolecular weight analysis.

TABLE 8 Data for the polymerization of MPEG(550)MA with initiator 8 at50° C. in 67 v/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 180 5 4743 4743 360 16 5904 5904 1320 65 11202 11202 1800 7812245 12245

[PEG]/[I]/[Cu]/[L]=64/1/1/2 in 67v/v % Toluene Solution at 50° C.

Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) andMPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (3.0 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-propyl-2-pyridylmethanimine(0.119 g, 0.80 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 9 Data for the polymerization of MPEG(550)MA with initiator 8 at50° C. in 67 v/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 180 13 4875 1.041 360 22 5601 1.087 1320 66 9897 1.091

[PEG]/[I]/[Cu]/[L]=6.4/1/2 in 67 v/v % Toluene Solution at 50° C.

Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) andMPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (3.0 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-octyl-2-pyridylmethanimine(0.175 g, 0.80 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 10 Data for the polymerization of MPEG(550)MA with initiator 8 at50° C. in 67 v/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 180 19 5034 1.075 360 33 6636 1.101 1320 85 11294 1.097

[PEG]/[I]/[Cu]/[L]=64/1/1/2 in 67 v/v % Toluene Solution at 70° C.

Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) andMPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (3.0 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.107 g, 0.80 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 70° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 11 Data for the polymerization of MPEG(550)MA with initiator 8 at70° C. in 67 v/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 60 20 5455 1.096 120 52 7898 1.114 180 73 9544 1.086 240 80 102071.095

[PEG]/[I]/[Cu]/[L]==6.4/1/112 in 67 v/v % Toluene Solution at 50° C.

Initiator 8 (6.0 g, 24 mmol), Cu(I)Br (3.44 g, 24 mmol, 1 eq) andMPEG(550)MA (96 g, 0.153 mol), and a magnetic follower were placed in anoven dried Schlenk tube. The Schlenk tube was evacuated and flushed withdry nitrogen three times. Deoxygenated toluene (176 mL) was added to theSchlenk tube. The resulting solution was deoxygenated by bubbling withnitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine (6.44g, 48 mmol) was added. The reaction was placed in a thermostaticallycontrolled oil bath at 50° C. (t=0) and samples were removedperiodically for conversion and molecular weight analysis. The polymerwas purified by removing the solvent in vacuo and dialysising theresidue using acidic water (pH ˜4). Subsequent freeze drying isolatedthe product.

TABLE 12 Data for the polymerization of MPEG(550)MA with initiator 8 at50° C. in 67 v/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 1200 66 8207 1.118 1500 75 9276 1.082 1680 81 9342 1.096

[PEG]/[I]/[Cu]/[L]==23.2/1/1/2 in 80 v/v % Toluene Solution at 50° C.

Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) andMPEG(1000)MA (10.0 g, 9.3 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (40 mL) was added tothe Schlenk tube. The resulting solution was deoxygenated by bubblingwith nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine(0.107 g, 0.80 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t—0) and samples wereremoved periodically for conversion and molecular weight analysis. Thepolymer was purified by removing the solvent in vacuo and dialysisingthe residue using acidic water (pH ˜4). Subsequent freeze dryingisolated the product.

TABLE 13 Data for the polymerization of MPEG(1000)MA with initiator 8 at50° C. in 80 w/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 300 3 9760 1.056 1260 41 19436 1.087 3000 79 29013 1.132 7020 8730046 1.149

[PEG]/[I]/[Cu]/[L]==46.3/1/1/2 in 80 v/v % Toluene Solution at 50° C.

Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) andMPEG(1000)MA (20.0 g, 18.5 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (80 mL) was added tothe Schlenk tube. The resulting solution was deoxygenated by bubblingwith nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine(0.107 g, 0.80 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis. Thepolymer was purified by removing the solvent in vacuo and dialysisingthe residue using acidic water (pH ˜4). Subsequent freeze dryingisolated the product.

TABLE 14 Data for the polymerization of MPEG(1000)MA with initiator 8 at50° C. in 80 w/v % toluene solution. Sample Conversion/ Time/minutes %Mn PDi 300 4 11014 1.070 1260 15 14388 1.080 3000 31 26378 1.096 7020 5333388 1.154

[PEG]/[I]/[Cu]/[L]==23.2/1/1/2 in 80 w/v % Toluene Solution at 50° C.

Initiator 8 (1.0 g, 4.0 mmol), Cu(I)Br (0.574 g, 4.0 mmol, 1 eq) andMPEG(1000)MA (100 g, 93.0 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (200 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated by bubblingwith nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine(1.07 g, 8.0 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis. Thepolymer was purified by removing the solvent in vacuo and dialysisingthe residue using acidic water (pH ˜4). Subsequent freeze dryingisolated the product.

TABLE 15 Data for the polymerization of MPEG(1000)MA with initiator 8 at50° C. in 80 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 4320 89 37676 1.143

Polymerisations Using Initiator 7

Polymerisation of MPEG(395)MA [PEG]/[I]/[Cu]/[L]=10/1/1/2 in 50 v/v %Toluene Solution at 30° C.

A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol),initiator 7 (0.601 g, 2.27 mmol) and a magnetic follower prior to beingdeoxygenated by cycling between nitrogen and vacuum three times. To asecond Schlenk tube was added MPEG(395)MA (10 mL, 22.74 mmol),N-(n-propyl)-2-pyridylmethanimine (0.71 mL, 4.54 mmol) and toluene (10mL). The mixture was immediately subjected to five freeze-pump-thawdegassing cycles. This solution was then transferred to the Schlenk tubecontaining the initiator and Cu(I)Br via a cannula. The resulting brownsolution was stirred at 30° C. Samples were removed periodically usingdegassed syringes and quenched in liquid nitrogen for conversion andmolecular weight analysis.

TABLE 16 Kinetic data for the polymerisation of MPEG(395)MA initiated by7 in toluene solution (50% v/v) at 30° C.([MPEG(395)MA]₀/[CuBr]₀/[NHSBr]₀/[ligand]₀ = 10/1/1/2). Solvent/ TimeConversion ln([M]₀/ M_(n,then) M_(n,SEC) Ligand (h) (%) [M]) (g · mol⁻¹)(g · mol⁻¹) M_(w)/M_(n) Toluene 1 8.9 0.0933 450 2350 1.10 Propyl 2 18.40.204 920 2860 1.26 ligand 3 27.1 0.316 1360 3100 1.20 4 34.7 0.42591740 3600 1.13 17 80.8 1.6510 4050 5670 1.06

[PEG]/[I]/[Cu]/[L]=10/1/1/2 in 50 v/v % Anisole Solution at 30° C.

A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol),initiator 7 (0.601 g, 2.27 mmol) and a magnetic follower prior to beingdeoxygenated by cycling between nitrogen and vacuum three times. To asecond Schlenk tube was added MPEG(395)MA (10 mL, 22.74 mmol),N-(n-ethyl)-2-pyridylmethanimine (0.64 mL, 4.54 mmol) and anisole (10mL). The mixture was immediately subjected to five freeze-pump-thawdegassing cycles. This solution was then transferred to the Schlenk tubecontaining the initiator and Cu(I)Br via a cannula. The resulting brownsolution was stirred at 30° C. Samples were removed periodically usingdegassed syringes and quenched in liquid nitrogen for conversion andmolecular weight analysis.

TABLE 17 Kinetic data for the polymerisation of MPEG(395)MA initiated by7 in anisole solution (50% v/v) at 30° C.([MPEG(395)MA]₀/[CuBr]₀/[NHSBr]₀/[ligand]₀ = 10/1/1/2). Solvent/ TimeConversion ln([M]₀/ M_(n, theo) M_(n, SEC) Ligand (h) (%) [M]) (g ·mol⁻¹) (g · mol⁻¹) M_(w)/M_(n) Anisole 2 17.0 0.1861 850 2670 1.11 Ethyl4 26.8 0.3116 1340 3460 1.12 ligand 6 34.8 0.4277 1740 4260 1.11 22 81.41.6809 4080 6350 1.06

[PEG]/[I]/[Cu]/[L]=10/1/1/2 in 50 v/v % Anisole Solution at 30° C.

A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol),initiator 7 (0.601 g, 2.27 mmol) and a magnetic follower prior to beingdeoxygenated by cycling between nitrogen and vacuum three times. To asecond Schlenk tube was added MPEG(395)MA (10 mL, 22.74 mmol),N-(n-propyl)-2-pyridylmethanimine (0.71 mL, 4.54 mmol) and anisole (10mL). The mixture was immediately subjected to five freeze-pump-thawdegassing cycles. This solution was then transferred to the Schlenk tubecontaining the initiator and Cu(I)Br via a cannula. The resulting brownsolution was stirred at 30° C. Samples were removed periodically usingdegassed syringes and quenched in liquid nitrogen for conversion andmolecular weight analysis.

TABLE 18 Kinetic data for the polymerisation of MPEG(395)MA initiated by7 in anisole solution (50% v/v) at 30° C.([MPEG(395)MA]₀/[CuBr]₀/[NHSBr]₀/[ligand]₀ = 10/1/1/2). Solvent/ TimeConversion ln([M]₀/ M_(n, theo) M_(n, SEC) Ligand (h) (%) [M]) (g ·mol⁻¹) (g · mol⁻¹) M_(w)/M_(n) Anisole 2 9.8 0.1030 490 2070 1.10 Propyl4 16.8 0.1837 842 2480 1.12 ligand 6 28.7 0.3378 1440 2870 1.13 27 83.41.7985 4180 6280 1.06

Polymerisation of MPEG(550)MA [PEG]/[I]/[Cu]/[L]=6.4/1/1/2 in 66 w/v %Toluene Solution at 30° C.

Initiator 7 (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) andMPEG(550)MA (7.57 g, 0.012 mol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (14 mL) was added tothe Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.51 g, 3.79 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 30° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 19 Data for the polymerization of MPEG(550)MA with initiator 7 at30° C. in 66 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 60 19 2850 1.04 131 32 3230 1.10 199 45 3560 1.12 250 53 37601.12 298 56 3980 1.12

[PEG]/[I]/[Cu]/[L]=6.4/1/1/2 in 66 w/v % Toluene Solution at 50° C.

Initiator 7 (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) andMPEG(550)MA) (7.57 g, 0.012 mol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (15 mL) was added tothe Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.51 g, 3.79 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 20 Data for the polymerization of MPEG(550)MA with initiator 7 at50° C. in 66 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 59 39 3212 1.09 126 56 3958 1.11 195 69 4375 1.13 246 75 46491.13 295 82 4874 1.13

[PEG]/[I]/[Cu]/[L]=23.9/1/1/2 in 66 w/v % Toluene Solution at 90° C.

Initiator 7 (2.5 g, 9.47 mmol), Cu(I)Br (1.35 g, 9.47 mmol, 1 eq) andMPEG(550)MA (142.0 g, 0.226 mol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (261 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-propyl-2-pyridylmethanimine(2.80 g, 0.019 mol) was added. The reaction was placed in athermostatically controlled oil bath at 90° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis Thepolymer was purified by the dropwise addition of the reaction solutionto a vigorously stirred solution of diethyl ether (1000 mL). Theresulting oil was washed with diethyl ether (3×1000 mL) and then driedin vacuo.

TABLE 21 Data for the polymerization of MPEG(550)MA with initiator 7 at90° C. in 66 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 48 21 4449 1.11 132 40 7198 1.08 185 44 7779 1.07 245 46 81051.09 300 48 8331 1.09

[PEG]/[I]/[Cu]/[L]=6.4/1/1/2 in 66 w/v % Toluene Solution at 50° C.

Initiator 7 (10.0 g, 0.038 mol), Cu(I)Br (5.41 g, 0.038 mol, 1 eq) andMPEG(550)MA (151.0 g, 0.240 mol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (302 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated by bubblingwith nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine(10.2 g, 0.0761 mol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0). Conversion wasfollowed by ¹H NMR spectrometry and molecular weight analysis by SEC.

TABLE 22 Data for the polymerization of MPEG(550)MA with initiator 7 at50° C. in 66 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 235 86.4 5140 1.13

[PEG]/[I]/[Cu]/[L]=6.46/1/1/2 in 62 w/v % Toluene at 50° C.

Initiator 7 (2.95 g, 1.119×10⁻² mol), Cu(I)Br (1.60 g, 1.119×10⁻² mol)and MPEG(550)MA (45.42 g, 7.23×10⁻² mol) and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Toluene (73 mL) was then added tothe Schlenk tube and the mixture degassed via three consecutive freeze,pump, thaw cycles. On completion deoxygenatedN-ethyl-2-pyridylmethanimine (3.16 mL, 2.24×10⁻² mol) was added and theSchlenk placed in a thermostatically controlled oil bath at 50° C. (t=0)and sampled for conversion and molecular weight analysis. The polymerwas isolated by washing with diethyl ether and subsequently dialysed inacidified water (pH ˜4)

TABLE 23 Data for the polymerization of MPEG(550)MA with initiator 7 at50° C. in 62 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 300 84.9 4590 1.22

[PEG]/[I]/[Cu]/[L]==23.9/1/1/2 in 67 w/v % Toluene Solution at 50° C.

Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq),2,2′-bipyridyl (0.059 g, 0.378 mmol), MPEG(550)MA, (2.84 g, 4.52 mmol)and a magnetic follower were placed in an oven dried Schlenk tube. TheSchlenk tube was evacuated and flushed with dry nitrogen three times.Deoxygenated toluene (5.68 mL) was added to the Schlenk tube and theresulting solution was deoxygenated via three freeze pump thaw cycles.The reaction was placed in a thermostatically controlled oil bath at 50°C. (t=0) and samples were removed periodically for conversion andmolecular weight analysis.

TABLE 24 Data for the polymerization of MPEG(550)MA with initiator 7 at50° C. in 67 w/v % toluene solution using 2,2′-bipyridyl ligand. SampleTime/ Conversion/ minutes % Mn PDi 240 85 15443 1.11

[PEG]/[I]/[Cu]/[L]=23.9/1/1/2 in 67 w/v % Toluene Solution at 50° C.

Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq),4,4′-dinonyl-2,2′-dipyridyl (0.1545 g, 0.378 mmol), MPEG(550)MA, (2.84g, 4.52 mmol) and a magnetic follower were placed in an oven driedSchlenk tube. The Schlenk tube was evacuated and flushed with drynitrogen three times. Deoxygenated toluene (5.68 mL) was added to theSchlenk tube and the resulting solution was deoxygenated via threefreeze pump thaw cycles. The reaction was placed in a thermostaticallycontrolled oil bath at 50° C. (t=0) and samples were removedperiodically for conversion and molecular weight analysis.

TABLE 25 Data for the polymerization of MPEG(550)MA with initiator 7 at50° C. in 67 w/v % toluene solution using 4,4′-dinonyl-2,2′-dipyridylligand. Sample Time/ Conversion/ minutes % Mn PDi 240 79 15936 1.16

[PEG]/[I]/[Cu]/[L]=23.9/1/1/1 in 67 w/v % Toluene Solution at 50° C.

Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq),1,1,4,7,10,10-hexamethyltriethylenetetramine (0.0435 g, 0.189 mmol),MPEG(550)MA, (2.84 g, 4.52 mmol) and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (5.68 mL) was addedto the Schlenk tube and the resulting solution was deoxygenated viathree freeze pump thaw cycles. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 26 Data for the polymerization of MPEG(550)MA with initiator 7 at50° C. in 67 w/v % toluene solution using1,1,4,7,10,10-hexamethyltriethylenetetramine ligand. Sample Time/Conversion/ minutes % Mn PDi 240 86 19060 1.16

[PEG]/[I]/[Cu]/[L]=23.9/1/1/1 in 67 w/v % Toluene Solution at 50° C.

Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq),N,N,N′,N″,N″-pentamethyldiethylenetriamine (0.0328 g, 0.189 mmol),MPEG(550)MA, (2.84 g, 4.52 mmol) and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (5.68 mL) was addedto the Schlenk tube and the resulting solution was deoxygenated viathree freeze pump thaw cycles. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 27 Data for the polymerization of MPEG(550)MA with initiator 7 at50° C. in 67 w/v % toluene solution usingN,N,N′,N″,N″-pentamethyldiethylenetriamine ligand. Sample Time/Conversion/ minutes % Mn PDi 240 95 19019 1.20

Polymerisation of MPEG(1000)MA [PEG]/[I]/[Cu]/[L]=13.9/1/1/2 in 66 w/v %Toluene Solution at 90° C.

Initiator 7 (0.526 g, 1.99 mmol), Cu(I)Br (0.29 g, 2.02 mmol, 1 eq) andMPEG(1000)MA (29.62 g, 0.027 mol), and a magnetic follower were placedin an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (60 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated viathree freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.51 g, 3.96 mol) was added. The reactionwas placed in a thermostatically controlled oil bath at 90° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis.

The polymer was purified by the dropwise addition of the reactionsolution to a vigorously stirred solution of diethyl ether (1000 mL).The resulting oil was washed with diethyl ether (3×1000 mL) and thendried in vacuo.

TABLE 28 Data for the polymerization of MPEG(1000)MA with initiator 7 at90° C. in 66 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 1250 47.3 12180 1.16 2460 50.4 12460 1.16 3890 52.8 12540 1.20

[PEG]/[I]/[Cu]/[L]=9.01/0.24/0.24 in 75 w/v % Toluene Solution at 50° C.

Initiator 7 (5.0 g, 0.019 mol), Cu(I)Br (0.66 g, 4.61 mmol, 0.24 eq) andMPEG(1000)MA (185.0 g, 0.171 mol), and a magnetic follower were placedin an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (740 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated bybubbling with nitrogen for 1 hour and then degassedN-ethyl-2-pyridylmethanimine (1.24 g, 9.24 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 50° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis.

TABLE 29 Data for the polymerization of MPEG(1000)MA with initiator 7 at50° C. in 75 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 60 7 5650 0.93 120 11 5595 0.97 285 20 6315 1.02 1340 43 79931.08 7476 63 9543 1.06

[PEG]/[I]/[Cu]/[L]=18.5/1/1/2 in 75 w/v % Toluene Solution at 50° C.

Initiator 7 (1.0 g, 3.79 mmol), Cu(I)Br (0.54 g, 3.79 mmol, 1 eq) andMPEG(1000)MA (151.4 g, 0.140 mol), and a magnetic follower were placedin an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (608 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated bybubbling with nitrogen for 1 hour and then degassedN-ethyl-2-pyridylmethanimine (1.02 g, 7.57 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 50° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis.

TABLE 30 Data for the polymerization of MPEG(1000)MA with initiator 7 at50° C. in 75 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 4005 100 8607 1.32

[PEG]/[I]/[Cu]/[L]=18.5/1/1/2 in 75 w/v % Toluene Solution at 50° C.

Initiator 7 (2.0 g, 7.57 mmol), Cu(I)Br (1.08 g, 7.57 mmol, 1 eq)MPEG(1000)MA (151.47 g, 0.140 mol), and a magnetic follower were placedin an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (606 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated bybubbling with nitrogen for 1 hour and then degassedN-ethyl-2-pyridylmethanimine (2.03 g, 0.015 mol) was added. The reactionwas placed in a thermostatically controlled oil bath at 50° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis.

TABLE 31 Data for the polymerization of MPEG(1000)MA with initiator 7 at50° C. in 75 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 195 20 7270 1.04 1380 53 11964 1.08 2735 73 13945 1.08

Polymerisation of MPEG(2000)MA

[PEG]/[I]/[Cu]/[L]=19.2/1/1/2 in 80 w/v % toluene solution at 30° C.

Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq)and (MPEG(2000)MA) (7.55 g, 3.63 mmol), and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (28 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated viathree freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.05 g, 0.38 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 30° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis. The polymer was purified by the dropwise addition of thereaction solution to a vigorously stirred solution of diethyl ether (400mL). The resulting white powder was filtered, dissolved in toluene (20mL) and precipitated in diethyl ether (400 mL). This procedure wasrepeated three times.

TABLE 32 Data for the polymerization of MPEG(2000)MA with initiator 7 at30° C. in 80 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 89 4 3380 1.04 291 9 9820 1.09 901 17 10030 1.07 1369 23 110801.07 2760 26 12610 1.07 3965 28 14830 1.04

[PEG]/[I]/[Cu]/[L]=19.2/1/1/2 in 80 w/v % Toluene Solution at 50° C.

Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq)and MPEG(2000)MA (7.55 g, 3.63 mmol), and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (28 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated viathree freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.05 g, 0.38 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 50° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis. The polymer was purified by the dropwise addition of thereaction solution to a vigorously stirred solution of diethyl ether (400mL). The resulting white powder was filtered, dissolved in toluene (20mL) and precipitated in diethyl ether (400 mL). This procedure wasrepeated three times.

TABLE 33 Data for the polymerization of MPEG(2000)MA with initiator 7 at50° C. in 80 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 86 7 8700 1.06 289 12 10920 1.07 899 24 14450 1.05 1367 33 158101.04 2758 45 20220 1.07 3962 53 23180 1.07

[PEG]/[I]/[Cu]/[L]=19.2/1/1/2 in 80 w/v % Toluene Solution at 90° C.

Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq)and MPEG(2000)MA (7.55 g, 3.63 mmol), and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (28 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated viathree freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.05 g, 0.38 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 90° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis. The polymer was purified by the dropwise addition of thereaction solution to a vigorously stirred solution of diethyl ether (400mL). The resulting white powder was filtered, dissolved in toluene (20mL) and precipitated in diethyl ether (400 mL). this procedure wasrepeated three times.

TABLE 34 Data for the polymerization of MPEG(2000)MA with initiator 7 at90° C. in 80 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 86 18 11100 1.08 289 26 14870 1.08 899 31 17900 1.08 1367 3518110 1.09 2758 38 18110 1.09 3962 39 18240 1.08

[PEG]/[I]/[Cu]/[L]==9.3/1/1/2 in 66 w/v % Toluene Solution at 90° C.

Initiator 7 (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) andMPEG(1000)MA (18.90 g, 0.018 mol), and a magnetic follower were placedin an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (35 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated viathree freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 90° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis.

TABLE 35 Data for the polymerization of MPEG(1000)MA with initiator 7 at90° C. in 66 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 4160 88.7 9870 1.22

[PEG]/[I]/[Cu]/[L]=19.2/1/1/2 in 80 w/v % Toluene Solution at 50/70° C.

Initiator 7 (0.67 g, 2.53 mmol), Cu(I)Br (0.36 g, 2.53 mmol, 1 eq) andMPEG(2000)MA (101.24 g, 0.049 mol), and a magnetic follower were placedin an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (405 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated viathree freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.68 g, 5.07 mmol) was added. The reactionwas placed in a thermostatically controlled oil bath at 50° C. (t=0) andsamples were removed periodically for conversion and molecular weightanalysis. The temperature was increased to 70° C. after 45 hours 15minutes.

TABLE 36 Data for the polymerization of MPEG(2000)MA with initiator 7 at50/70° C. in 80 w/v % toluene solution. Sample Time/ Conversion/ minutes% Mn PDi 4030 47 24,600 1.06

[PEG]/[I]/[Cu]/[L]=19.2/1/1/2 in 75 w/v % Toluene at 50/70° C.

Initiator 7 (0.66 g, 2.5×10⁻³ mol), Cu(I)Br (0.36 g, 2.5×10⁻³ mol) andMPEG(2000)MA (100.0 g, 4.81×10⁻² mol) and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Toluene (300 mL) was then addedto the Schlenk tube and the mixture deoxygenated by purging withnitrogen for 1 hour. Deoxygenated N-ethyl-2-pyridylmethanimine (0.706mL, 5.0×10⁻³ mol) was then added and the Schlenk placed in athermostatically controlled oil bath at 50° C. (t=0). The temperaturewas increased to 70° C. after 24 hours. The polymer was isolated bywashing with diethyl ether and subsequently dialysed in acidified water(pH ˜4).

TABLE 37 Data for the polymerization of MPEG(2000)MA with initiator 7 at50/70° C. in 75 w/v % toluene solution. Sample Time/ Conversion/ minutes% Mn PDi 2880 94.2 21900 1.21

[PEG]/[I]/[Cu]/[L]=28.8/1/1/2 in 75 w/v % Toluene at 50/70° C.

Initiator 7 (0.44 g, 1.67×10⁻³ mol), Cu(I)Br (0.24 g, 1.67×10⁻³ mol) andMPEG(2000)MA (100.0 g, 4.81×10⁻² mol) and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Toluene (300 mL) was then addedto the Schlenk tube and the mixture deoxygenated by purging withnitrogen for 1 hour. Deoxygenated N-ethyl-2-pyridylmethanimine (0.47 mL,3.33×10⁻³ mol) was then added and the Schlenk placed in athermostatically controlled oil bath at 50° C. C (t=0). The temperaturewas increased to 70° C. after 24 hours. The polymer was isolated bywashing with diethyl ether and subsequently dialysed in acidified water(pH ˜4).

TABLE 38 Data for the polymerization of MPEG(2000)MA with initiator 7 at50/70° C. in 75 w/v % toluene solution. Sample Time/ Conversion/ minutes% Mn PDi 2880 92.8 26370 1.26

Polymerisations Using Initiator 5

Polymerisation of MPEG(550)MA [PEG]/[I]/[Cu]/[L]=64/1/in 75 w/v %Toluene Solution at 50/90° C.

Initiator 5 (0.25 g, 0.622 mmol), Cu(I)Br (0.089 g, 0.622 mmol, 1 eq)and MPEG(550)MA (24.90 g, 0.040 mol), and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (100 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated bybubbling with nitrogen for 1 hour and then degassedN-ethyl-2-pyridylmethanimine (0.167 g, 1.245 mmol) was added. Thereaction was placed in a thermostatically controlled oil bath at 50° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis. The temperature was increased to 90° C. after 3 hours15 minutes.

TABLE 39 Data for the polymerization of MPEG(550)MA with initiator 5 at50/90° C. in 75 w/v % toluene solution. Sample Time/ Conversion/ minutes% Mn PDi 150 3 5376 1.07 353 9 10390 1.09 1750 27 13890 1.16

Polymerisation of MPEG(1000)MA [PEG]/[I]/[Cu]/[L]=37/1/1/2 in 75 w/v %Toluene Solution at 50° C.

Initiator 5 (0.125 g, 0.031 mmol), Cu(I)Br (0.044 g, 0.031 mmol, 1 eq)and MPEG(1000)MA (12.45 g, 0.012 mol), and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (50 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated bybubbling with nitrogen for 1 hour and then degassedN-ethyl-2-pyridylmethanimine (0.083 g, 0.062 mmol) was added. Thereaction was placed in a thermostatically controlled oil bath at 50° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis.

TABLE 40 Data for the polymerization of MPEG(1000)MA with initiator 5 at50° C. in 75 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 62 0 0 0 352 10 9713 1.06 1716 15 10924 1.08 2725 16 11240 1.084142 15 12800 1.11

Polymerisations Using Initiator 9

Polymerisation of MPEG(2000)MA [PEG]/[I]/[Cu]/[L]=28.8:1/1/2 in 67 w/v %Acetone at 50° C.

Initiator 9 (0.035 g, 1.667×10⁻⁴ mol), Cu(I)Br (0.024 g, 1.667×10⁻⁴ mol)and MPEG(2000)MA (10 g, 4.81×10⁻³ mol) and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Acetone (20 mL) was then added tothe Schlenk tube and the mixture degassed via three consecutive freeze,pump, thaw cycles. On completion deoxygenatedN-ethyl-2-pyridylmethanimine (0.05 mL, 3.54×10⁻⁴ mol) was added and theSchlenk placed in a thermostatically controlled oil bath at 50° C. (t=0)and samples removed periodically for conversion and molecular weightanalysis.

TABLE 41 Data for the polymerization of MPEG(2000)MA with initiator 9 at50° C. in 67 w/v % acetone solution Sample Time/ Conversion/ minutes %Mn PDi 60 3 25270 1.06 360 19 22690 1.08 3480 74 32080 1.14

Polymerisations Using Initiator 6

Polymerisation of MPEG(2000)MA [PEG]/[I]/[Cu]/[L]=14.4/1/1/2 in 67 w/v %Toluene at 30° C.

Initiator 6 (0.119 g, 3.333×10⁻⁴ mol), Cu(I)Br (0.048 g, 3.333×10⁻⁴ mol)and MPEG(2000)MA (10 g, 4.81×10⁻³ mol) and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Toluene (20 mL) was then added tothe Schlenk tube and the mixture degassed via three consecutive freeze,pump, thaw cycles. On completion deoxygenatedN-n-propyl-2-pyridylmethanimine (0.10 mL, 6.667×10⁻⁴ mol) was added andthe Schlenk placed in a thermostatically controlled oil bath at 30° C.(t=0) and sampled for conversion and molecular weight analysis.

TABLE 42 Data for the polymerization of MPEG(2000)MA with initiator 6 at30° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 3900 29.5 24120 1.08

[PEG]/[I]/[Cu]/[L]=21.63/1/1/2 in 67 w/v % Toluene at 30° C.

Initiator 6 (0.079 g, 2.222×10⁻⁴ mol), Cu(I)Br (0.031 g, 2.222×10⁻⁴ mol)and PEG(2000)MA (10 g, 4.81×10⁻³ mol) and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Toluene (20 mL) was then added tothe Schlenk tube and the mixture degassed via three consecutive freeze,pump, thaw cycles. On completion deoxygenatedN-n-propyl-2-pyridylmethanimine (0.066 mL, 4.444×10⁻⁴ mol) was added andthe Schlenk placed in a thermostatically controlled oil bath at 30° C.(t=0) and sampled for conversion and molecular weight analysis.

TABLE 43 Data for the polymerization of MPEG(2000)MA with initiator 6 at30° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 6600 27 27480 1.10

[PEG]/[I]/[Cu]/[L]=28.8/1/1/2 in 67 w/v % Toluene at 30° C.

Initiator 6 (0.059 g, 1.667×10⁻⁴ mol), Cu(I)Br (0.024 g, 1.667×10⁻⁴ mol)and MPEG(2000)MA (10 g, 4.81×10⁻³ mol) and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Toluene (20 mL) was then added tothe Schlenk tube and the mixture degassed via three consecutive freeze,pump, thaw cycles. On completion deoxygenatedN-n-propyl-2-pyridylmethanimine (0.049 mL, 1.667×10⁻⁴ mol) was added andthe Schlenk placed in a thermostatically controlled oil bath at 30° C.(t=0) and sampled for conversion and molecular weight analysis.

TABLE 44 Data for the polymerization of MPEG(2000)MA with initiator 6 at30° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 6600 18.3 25290 1.09

Polymerisations Using Initiator 10

Polymerisation of MPEG(395)MA [PEG]/[I]/[Cu]/[L]==25/1/1/2 in 67 w/v %Toluene at 50° C.

Initiator 10 (0.81 g, 1.68×10⁻³ mol), Cu(I)Br (0.24 g, 1.68×10⁻³ mol)and MPEG(395)MA (20.0 g, 4.21×10−2 mol) and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Toluene (41 mL) was then added tothe Schlenk tube and the mixture deoxygenated by purging with nitrogenfor 1 hour. Deoxygenated N-n-propyl-2-pyridylmethanimine (0.53 mL,3.37×10⁻³ mol) was then added and the Schlenk placed in athermostatically controlled oil bath at 50° C. (t=0) and sampled forconversion and molecular weight analysis.

TABLE 45 Data for the polymerization of MPEG(395) with initiator 10 at50° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 900 52.9 6300 1.11Polymerisations using Initiator 11

Polymerisation of MPEG(550)MA [PEG]/[I]/[Cu]/[L]=6.4/1/1/2 in 67 v/v %Toluene Solution at 50° C.

Initiator 11 (0.103 g, 0.380 mmol), Cu(I)Br (0.054 g, 0.380 mmol, 1 eq)and MPEG(550)MA (1.51 g, 2.41 mmol), and a magnetic follower were placedin an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (2.78 mL)was added to the Schlenk tube. The resulting solution was deoxygenatedvia three freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.10 g, 0.758 mmol) was added. Thereaction was placed in a thermostatically controlled oil bath at 50° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis.

TABLE 46 Data for the polymerization of MPEG(550)MA with initiator 11 at50° C. in 67 v/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 60 24 3545 1.080 120 41 4002 1.102 180 53 4243 1.104 240 64 45061.109 300 70 4677 1.108

[PEG]/[I]/[Cu]/[L]=6.4/1/1/2 in 67 v/v % Toluene Solution at 50° C.

Initiator 11 (3.0 g, 11.1 mmol), Cu(I)Br (1.584 g, 11.1 mmol, 1 eq) andMPEG(550)MA (44.27 g, 70.5 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (81.3 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated by bubblingwith nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine(2.97 g, 22.2 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis. Thepolymer was purified by removing the solvent in vacuo and dialysisingthe residue using acidic water (pH ˜4). Subsequent freeze dryingisolated the product.

TABLE 47 Data for the polymerization of MPEG(550)MA with initiator 11 at50° C. in 67 v/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 60 27 4800 1.096 120 45 5427 1.115 180 60 5895 1.127 240 67 62151.133 300 72 6343 1.125

Polymerisation of MPEG(2000)MA [PEG]/[I]/[Cu]/[L]=12/11/2 in 80 w/v %Toluene Solution at 50/70° C.

Initiator 11 (0.1 g, 0.369 mmol), Cu(I)Br (0.053 g, 0.369 mmol, 1 eq)and MPEG(2000)MA (9.24 g, 4.44 mmol), and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (37 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated bybubbling with nitrogen for 1 hour and then degassedN-ethyl-2-pyridylmethanimine (0.10 g, 0.758 mmol) was added. Thereaction was placed in a thermostatically controlled oil bath at 50° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis. The temperature was increased to 70° C. after 113hours. The polymer was purified by removing the solvent in vacuo anddialysising the residue using acidic water (pH ˜4). Subsequent freezedrying isolated the product.

TABLE 48 Data for the polymerization of MPEG(2000)MA with initiator 11at 50/70° C. in 80 w/v % toluene solution. Sample Time/ Conversion/minutes % Mn PDi 1020 21 — — 1440 23 — — 6780 40 — — 9660 62 20333 1.117

[PEG]/[I]/[Cu]/[L]=24/1/1/2 in 80 w/v % Toluene Solution at 50/70° C.

Initiator 11 (0.1 g, 0.369 mmol), Cu(I)Br (0.053 g, 0.369 mmol, 1 eq)and MPEG(2000)MA (18.48 g, 8.88 mmol), and a magnetic follower wereplaced in an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (74 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated bybubbling with nitrogen for 1 hour and then degassedN-ethyl-2-pyridylmethanimine (0.10 g, 0.758 mmol) was added. Thereaction was placed in a thermostatically controlled oil bath at 50° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis. The temperature was increased to 70° C. after 113hours. The polymer was purified by removing the solvent in vacuo anddialysising the residue using acidic water (pH ˜4). Subsequent freezedrying isolated the product.

TABLE 49 Data for the polymerization of MPEG(2000)MA with initiator 11at 50/70° C. in 80 w/v % toluene solution. Sample Time/ Conversion/minutes % Mn PDi 1020 16 — — 2580 19 6780 22 — — 8220 53 — — 9660 0.5722262 1.140

Polymerisations Using Initiator 12

Polymerisation of MPEG(1000)MA [PEG]/[I]/[Cu]/[L]=5/1/1/2 in 66 v/v %Toluene Solution at 70° C.

N-(ethyl)-2-pyridylmethanimine ligand (1.41 mL, 10.92 mmol), initiator12 (1.633 g, 5.46 mmol), and MPEG(1000)MA (27.3 mL, 30 g, 27.3 mmol)were charged to a dry Schlenk tube along with toluene (60 mL) as thesolvent and mesitylene (1 mL) as an internal standard. The tube wassealed with a rubber septum and subjected to three freeze pump thawcycles. This solution was then cannulated under nitrogen into anotherSchlenk tube, previously evacuated and filled with nitrogen, containingCu(I)Cl (0.543 g, 5.46 mmol) and a magnetic follower. The brown solutionwas subsequently heated to 70° C. with constant stirring (t=0). Sampleswere removed periodically using a degassed syringe for molecular weightand conversion analysis. After 48 h the mixture was diluted with 50 mLof toluene, air was bubbled for 6 h and the green suspension was kept at0° C. overnight. After filtration through a short neutral alumina columnto remove the copper salt, the polymer was precipitated from diethylether. The polymer was collected by filtration and dried in vacuum oven(40° C.) overnight.

TABLE 50 Data for the polymerization of MPEG(1000)MA with initiator 12at 70° C. in 66 v/v % toluene solution. Sample Time/ Conversion/ hours %Mn PDi 1 17.8 1370 1.23 2.5 56.6 10700 1.09 4 73.5 11600 1.15 5 78.911600 1.11 6 81.3 11600 1.19 7 85.9 12600 1.15

[PEG]/[I]/[Cu]/[L]=20/1/1/2 in 66 v/v % Toluene Solution at 50° C.

N-(ethyl)-2-pyridylmethanimine ligand (0.35 mL, 2.73 mmol), initiator 12(0.41 g, 1.37 mmol), PEG(1000)MA (27.3 mL, 30 g, 27.3 mmol) were chargedto a dry Schlenk tube along with toluene (60 mL) as the solvent andmesitylene (1 mL) as internal standard. The tube was sealed with arubber septum and subjected to three freeze pump thaw cycles. Thissolution was then cannulated under nitrogen into another Schlenk tube,previously evacuated and filled with nitrogen, containing Cu(I)Br (0.197g, 1.37 mmol) and a magnetic follower. The brown solution wassubsequently heated to 50° C. with constant stirring (t=0). Samples wereremoved periodically using a degassed syringe for molecular weight andconversion analysis. Half the reaction solution was removed with a drycannula when conversion was at 66%, bubbled for 6 h with air, and passedover a short neutral alumina column to removed copper salt. The solventwas removed under vacuum and the unreacted monomer was removed bydialysis to give the polymer as a white powder. After 48 h the remainingreaction mixture was diluted with 50 mL of toluene, air was bubbled for6 h and the green suspension was kept at 0° C. overnight. Afterfiltration through a short neutral alumina column to remove the coppersalt, the polymer was precipitated from diethyl ether. The polymer wascollected by filtration and dried in vacuum oven (40° C.) overnight.

TABLE 51 Data for the polymerization of MPEG(1000)MA with initiator 12at 50° C. in 66 v/v % toluene solution. Sample Time/ Conversion/ hours %Mn PDi 1 5 1566 1.14 3 11 11400 1.06 6 26 13000 1.07 8 31 12700 1.09 2161 13600 1.13 24 66 13800 1.14 28 73 14500 1.2 31 76 14900 1.21 46 8714600 1.18 50.5 89 16000 1.2 54 0.9 17600 1.19

Polymerisations Using Initiator 13

Polymerisation of MPEG(550)MA [PEG]/[I]/[Cu]/[L]=15.9/1/1/2 in 67 w/v %Toluene Solution at 30° C.

Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) andMPEG(550)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (5.5 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.074 g, 0.56 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 30° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 52 Data for the polymerization of MPEG(550)MA with initiator 13 at30° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 1440 7.8 5626 1.08 4260 14.0 6153 1.11

[PEG]/[I]/[Cu]/[L]=8/1/1/2 in 67 w/v % Toluene Solution at 50° C.

Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) andMPEG(550)MA (1.38 g, 2.20 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (2.75 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.074 g, 0.56 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 53 Data for the polymerization of MPEG(550)MA with initiator 13 at50° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 7200 44.8 7880 1.16

[PEG]/[I]/[Cu]/[L]=15.9/1/1/2 in 67 w/v % Toluene Solution at 50° C.

Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) andMPEG(550)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (5.5 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.074 g, 0.56 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 54 Data for the polymerization of MPEG(550)MA with initiator 13 at50° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 1440 16.4 7971 1.12 8640 27.2 8378 1.14

[PEG]/[I]/[Cu]/[L]=31.8/1/1/2 in 67 w/v % Toluene Solution at 50° C.

Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) andMPEG(550)MA (5.51 g, 8.77 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (11.0 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.074 g, 0.56 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 50° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 55 Data for the polymerization of MPEG(550)MA with initiator 13 at50° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 1440 9.6 8504 1.11 5760 16.8 9999 1.14 8640 17.8 10208 1.13

[PEG]/[I]/[Cu]/[L]=15.9/1/1/2 in 67 w/v % Toluene Solution at 70° C.

Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) andMPEG(550)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed inan oven dried Schlenk tube. The Schlenk tube was evacuated and flushedwith dry nitrogen three times. Deoxygenated toluene (5.5 mL) was addedto the Schlenk tube. The resulting solution was deoxygenated via threefreeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine(0.074 g, 0.56 mmol) was added. The reaction was placed in athermostatically controlled oil bath at 70° C. (t=0) and samples wereremoved periodically for conversion and molecular weight analysis.

TABLE 56 Data for the polymerization of MPEG(550)MA with initiator 13 at70° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes %Mn PDi 120 13.6 5768 1.09 300 21.3 6814 1.10 4260 41.0 8444 1.15

[PEG]/[I]/[Cu]/[L]=15.9/1/1/2 in 67 w/v % Toluene Solution at 50/90° C.

Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Cl (0.0273 g, 0.28 mmol, 1 eq)and MPEG(550)MA (2.76 g, 4.39 mmol), and a magnetic follower were placedin an oven dried Schlenk tube. The Schlenk tube was evacuated andflushed with dry nitrogen three times. Deoxygenated toluene (5.5 mL) wasadded to the Schlenk tube. The resulting solution was deoxygenated viathree freeze pump thaw cycles and then degassedN-ethyl-2-pyridylmethanimine (0.074 g, 0.56 mmol) was added. Thereaction was placed in a thermostatically controlled oil bath at 50° C.(t=0) and samples were removed periodically for conversion and molecularweight analysis. The temperature was increased to 90° C. after 163 hours

TABLE 57 Data for the polymerization of MPEG(550)MA with initiator 13 at50/90° C. in 67 w/v % toluene solution. Sample Time/ Conversion/ minutes% Mn PDi 9780 3.8 4090 1.09 12660 81.0 16504 1.31

Polymerisations Using Initiator 14

Polymerisation of MPEG(395)MA [PEG]/[I]/[Cu]/[L]=6/1/1/2 in 50 v/v %Toluene Solution at 40° C.

N-(ethyl)-2-pyridylmethanimine ligand (1.07 mL, 1.017 g, 7.58×10⁻³ mol),initiator 14 (1.229 g, 3.79×10⁻³ mol) and MPEG(395)MA (10.80 g,22.70×10⁻³ mol) were charged to a dry Schlenk tube along with toluene(10 mL) as the solvent (50% v/v). The tube was sealed with a rubberseptum and subjected to three freeze-pump-thaw cycles. This solution wasthen cannulated under nitrogen into another Schlenk tube, previouslyevacuated and filled with nitrogen, containing Cu(I)Br (0.544 g,3.79×10⁻³ mol) and a magnetic follower. The brown solution wassubsequently heated to 40° C. with constant stirring (t=0). Samples wereremoved periodically using a degassed syringe for molecular weight andconversion analysis. After 48 h the mixture was diluted with 50 mL oftoluene, air was bubbled for 6 h and the green suspension was kept at 0°C. overnight. After filtration through a Celite® pad, the solvent wasremoved under reduced pressure to give a yellow-brown oil which wasdissolved in water (250 mL) and purified by dialysis (Millipore,regenerated cellulose, MWCO 1 kDa, filtration area 0.23 m²) to give theexpected polymer as a pale yellow oil.

TABLE 58 Polymerisation data for TMM-LRP of MPEG(395)MA using initiator14, [monomer]:[initiator]:[CuBr]:[L] = 6:1:1:2. Monomer/ Time Conv.Initiator (mins) (%) ln([M]₀/[M]) M_(n) PDI 6:1 (40° C.) 60 34.76 0.4785876 1.03 120 49.43 0.667 7250 1.08 180 62.05 0.889 8089 1.08 240 71.541.118 9013 1.10 300 76.33 1.359 9262 1.10 360 80.18 1.556 9561 1.07 42086.93 1.904 9932 1.10 480 88.72 2.216 10195 1.10

[PEG]/[I]/[Cu]/[L]=28/1/1/2 in 50 v/v % Toluene Solution at 40° C.

N-(ethyl)-2-pyridylmethanimine ligand (1.07 mL, 1.017 g, 7.58×10⁻³ mol),initiator 14 (0.263 g, 0.812×10⁻³ mol) and MPEG(395)MA (10.80 g,22.70×10⁻³ mol) were charged to a dry Schlenk tube along with toluene(10 mL) as the solvent (50% v/v). The tube was sealed with a rubberseptum and subjected to three freeze-pump-thaw cycles. This solution wasthen cannulated under nitrogen into another Schlenk tube, previouslyevacuated and filled with nitrogen, containing Cu(I)Br (0.116 g,0.812×10⁻³ mol) and a magnetic follower. The brown solution wassubsequently heated to 40° C. with constant stirring (t=0). Samples wereremoved periodically using a degassed syringe for molecular weight andconversion analysis. After 48 h the mixture was diluted with 50 mL oftoluene, air was bubbled for 6 h and the green suspension was kept at 0°C. overnight. After filtration through a Celite® pad, the solvent wasremoved under reduced pressure to give a yellow-brown oil which wasdissolved in water (250 mL) and purified by dialysis (Millipore,regenerated cellulose, MWCO 1 kDa, filtration area 0.23 m²) to give theexpected polymer as a pale yellow oil.

TABLE 59 Polymerisation data for TMM-LRP of MPEG(395)MA using initiator14, [monomer]:[initiator]:[CuBr]:[L] = 28:1:1:2. T = 40° C. Monomer/Time Conv. Initiator (mins) (%) ln([M]₀/[M]) M_(n) PDI 28:1 (40° C.) 6021.8 0.246 5500 1.05 120 24.7 0.284 5798 1.06 180 37.4 0.468 7001 1.09240 41.5 0.536 7202 1.08 300 46.2 0.620 7633 1.07 360 49.3 0.680 77331.09 420 50.7 0.707 7899 1.09 480 55.4 0.808 8099 1.08

[PEG]/[I]/[Cu]/[L]=28/1/1/2 in 50 v/v % Toluene Solution at 60° C.

N-(ethyl)-2-pyridylmethanimine ligand (1.07 mL, 1.02 g, 7.58×10⁻³ mol),initiator 14 (0.263 g, 0.812×10⁻³ mol) and MPEG(395)MA (10.80 g,22.70×10⁻³ mol) were charged to a dry Schlenk tube along with toluene(10 mL) as the solvent (50% v/v). The tube was sealed with a rubberseptum and subjected to three freeze-pump-thaw cycles. This solution wasthen cannulated under nitrogen into another Schlenk tube, previouslyevacuated and filled with nitrogen, containing Cu(I)Br (0.116 g,0.812×10⁻³ mol) and a magnetic follower. The brown solution wassubsequently heated to 60° C. with constant stirring (t=0). Samples wereremoved periodically using a degassed syringe for molecular weight andconversion analysis. After 48 h the mixture was diluted with 50 mL oftoluene, air was bubbled for 6 h and the green suspension was kept at 0°C. overnight. After filtration through a Celite® pad, the solvent wasremoved under reduced pressure to give a yellow-brown oil which wasdissolved in water (250 mL) and purified by dialysis (Millipore,regenerated cellulose, MWCO 1 kDa, filtration area 0.23 m²) to give theexpected polymer as a pale yellow oil.

TABLE 60 Polymerisation data for TMM-LRP of MPEG(395)MA using initiator14, [monomer]:[initiator]:[CuBr]:[L] = 28:1:1:2. T = 60° C. Monomer/Time Conv. Initiator (mins) (%) ln([M]₀/[M]) M_(n) PDI 28:1 (60° C.) 6038.0 0.427 5233 1.06 120 48.7 0.682 5656 1.07 180 58.9 0.969 6116 1.06240 67.3 1.257 6185 1.08 300 74.3 1.441 6416 1.07 360 78.9 1.618 62841.08 420 85.1 2.035 6291 1.08 480 89.1 2.182 6610 1.08[PEG]/[I]/[Cu]/[L]=10/1/1/2 in 50 v/v % d₈-Toluene Solution at 40° C.

N-(n-octyl)-2-pyridylmethanimine ligand (0.052 mL, 0.050 g, 0.228×10⁻³mol), initiator 14 (0.037 g, 0.114×10⁻³ mol) and MPEG(395)MA (0.050 mL,0.540 g, 1.14×10⁻³ mol) were charged to a dry Schlenk tube along withd₈-toluene (0.50 mL) as the solvent (50% v/v). The tube was sealed witha rubber septum and subjected to three freeze-pump-thaw cycles. Thissolution was then cannulated under nitrogen into an NMR tube, previouslyevacuated and filled with nitrogen, containing Cu(I)Br (0.016 g,0.114×10⁻³ mol). The tube was then heated to 40° C. and ¹H NMR spectrawere recorded every 15 minutes.

Polymerisations Using Initiator 15

Polymerisation of MPEG(395)MA [PEG]/[I]/[Cu]/[L]=8/1/1/2 in 50 wv %Toluene Solution at 30° C.

N-(ethyl)-2-pyridylmethanimine ligand (0.80 mL, 0.76 g, 5.68×10⁻³ mol),initiator 15 (2.03 g, 5.68×10⁻³ mol) MPEG(395)MA (20.0 mL, 21.6 g,45.50×10⁻³ mol) were charged to a dry Schlenk tube along with toluene(20 mL) as the solvent (50% v/v). The tube was sealed with a rubberseptum and subjected to three freeze-pump-thaw cycles. This solution wasthen cannulated under nitrogen into another Schlenk tube, previouslyevacuated and filled with nitrogen, containing Cu(I)Br (0.41 g,2.84×10⁻³ mol) and a magnetic follower (t=0). The brown solution wassubsequently stirred at 30° C. Samples were removed periodically using adegassed syringe for molecular weight and conversion analysis. After 7 hthe mixture was diluted with 50 mL of toluene, air was bubbled for 6 hand the green suspension was kept at 0° C. overnight. After filtrationthrough a Celite® pad, the solvent was removed under reduced pressure togive a yellow-brown oil which was dissolved in water (250 mL) andpurified by dialysis (Millipore, regenerated cellulose, MWCO I kDa,filtration area 0.23 m²) to give the expected polymer as a pale yellowoil.

TABLE 61 Polymerisation data for TMM-LRP of MPEG(395)MA using initiator15, [monomer]:[initiator]:[CuBr]:[L] = 8:1:1:2. T = 30° C. Monomer/ TimeConv. Initiator (mins) (%) ln([M]₀/[M]) M_(n) PDI 8:1 (30° C.) 60 22.900.478 4532 1.07 120 34.64 0.667 5008 1.11 180 45.66 0.889 5379 1.11 24650.97 1.118 5662 1.09 420 67.14 1.359 6165 1.08

Reactions of PolyPEG Polymers

Reactions of PolyPEG Polymers Prepared from Initiator 8

Hydrolytic Stability of the Succinimide End Group of PolyPEG PolymerInitiated by 8

On-line ¹H NMR experiments was carried out, each using a differentbuffer. N-succinimidyl (initiator 8) terminated poly(MPEG(395)MA(Mn=6400 g/mol, PDI=1.09) (50 mg, 0.00781×10⁻³ mol) were introduced inan NMR tube and dissolved in 0.5 mL of the appropriate phosphate buffer(pH=8, C=100 mM or 200 mM). NMR spectra were recorded regularly.

TABLE 62 Kinetic data for the hydrolysis of N-succinimidyl terminatedpoly(MPEG(395)MA initiated by 8 in different buffers. 100 mM phosphate200 mM phosphate buffer (pH = 8) buffer (pH = 8) Time Conversion TimeConversion (h) (%) (h) (%) 0 0 0 0 0.5 4.8 0.5 8.6 1 9.5 1 13.5 1.5 10.11.5 17.5 2 15.8 2 19.8 2.5 14.2 2.5 22.7 3 15.1 3 25.5 3.5 18.5 3.5 26.74 22.4 4 28.9 4.5 23.2 4.5 33.7 5 26.0 5 33.2 5.5 30.1 5.5 37.1 6 28.7 638.9 6.5 30.7 6.5 39.6 7 30.7 7 44.1 7.5 30.1 7.5 43.0 8 37.3 8 44.0 8.535.9 8.5 45.2 9 36.7 9 45.8 9.5 40.7 9.5 46.5 10 45.2 10 50.6 10.5 40.810.5 49.7 11 43.2 11 52.2 11.5 42.5 11.5 52.5 12 44.8 12 55.6 12.5 45.412.5 55.5 13 45.1 13 56.9 13.5 45.0 13.5 53.7 14.5 48.9 15 48.4 15.550.1 16 49.6 16.5 50.1 17 52.6 17.5 50.2 18 53.2 18.5 52.0 19 54.9 19.555.1 20 53.9 20.5 55.5 21 55.9 21.5 53.0 22 54.5 22.5 55.9 23 55.3 23.557.6 24 58.9

Bioconjuction of Succinimide Terminated PolyPEG Polymer Initiated by 8

A set of three experiments was carried out, each containing a differentratio polymer/lysozyme. Low molecular weight succinimidyl esterterminated poly(MPEG(395)MA) prepared from initiator 8 (Mn=6400 g/mol,PDI=1.11) (8.9 mg, 1.39×10⁻⁶ mol) for a ratio 2/1, (22.6 mg, 3.50×10⁻⁶mol) for a ratio 5/1 and (89.5 mg, 13.99×10⁻⁶ mol) for a ratio 20/1 andlysozyme (10 mg, 0.699×10⁻⁶ mol) was dissolved in 10 ml of anhydrousDMSO and 0.5 mL of anhydrous TEA and stirred at room temperature undernitrogen. Samples were taken periodically and analyzed by HPLC. The HPLCsystem was fitted with a guard column, a BioSep-SEC-S3000 column and aUV detector continuously measuring the relative absorbance of the mobilephase at 215 nm. The system was eluted with 0.1% v/v trifluoroaceticacid solution in water and acetonitrile (69/31 v/v) at a rate of 0.5mL/min. In the case of a ratio 30:1, the crude was analysed by SDS-PAGE(polyacrylamide resolving gel cross-linking: 15%, running buffer. 25 mMTRIS base, 250 mM glycine, 0.1% SDS, pH 8.7).

Reactions of PolyPEG Polymers Prepared from Initiator 7

Hydrolytic Stability of the Succinimide End Group of PolyPEG PolymerInitialed by 7

On-line 1H NMR experiments was carried out, each using a differentbuffer. N-succinimidyl (initiator 7) terminated Poly(MPEG(395)MA(Mn=2700 g/mol, PDI=1.12) (50 mg, 0.0185×10-3 mol) were introduced in anNMR tube and dissolved in 0.5 mL of the appropriate buffer (200 mMphosphate buffer (pH=6 and pH=8), 100 mM phosphate buffer (pH=8) or 200mM borate buffer (pH=9.2)). NMR spectra were recorded regularly.

TABLE 63 Kinetic data for the hydrolysis of the succinimide end group ofPoly(MPEG(395)MA polymer initiated by 7 in different buffers. 200 mM 100mM 200 mM 200 mM phosphate phosphate phosphate borate buffer bufferbuffer buffer (pH = 6) (pH = 8) (pH = 8) (pH = 9.2) Con- Con- Con- Con-Time version Time version Time version Time version (h) (%) (h) (%) (h)(%) (h) (%) 0.03 5.0 0.5 2.5 0.5 3 48 0.8 0.067 15.0 1 4.8 1 5.48 192 30.10 25.0 1.5 6.4 1.5 7.6 336 5.2 0.13 35.0 2 9.1 2 9.13 504 9.6 0.1743.8 2.5 10.2 3 11.56 0.20 48.5 3 11.2 4 14.33 0.23 55.0 3.5 12.4 5 16.50.27 60.0 4 14.1 21 31.7 0.30 64.0 4.5 16.1 22 31.32 0.33 66.0 5 17.4 2333.86 0.37 69.0 5.5 19.6 24 34.04 0.40 70.0 6 20.6 25 33.35 0.43 72.06.5 24 39 42.73 0.47 73.0 7 26.2 57 49.2 0.50 74.5 7.5 26.9 77 54.110.67 81.4 8 27.7 99 57.3 0.83 87.2 8.5 29.4 125 63 1.00 92.9 9 28.9 14665.24 1.17 96.7 9.5 30 171 67.35 1.33 100 10 32.7 195 69.7 12.5 35.7 22072.22 15 38.9 257 75 17.5 41.9 270 75.47 20 46 299 78.07 22.5 49 25 50.727.5 53.4 30 54.4 32.5 57.6 35 59 37.5 61.6 40 62.9 42.5 65.1 45 66.347.5 68.4 50 69.1 52.5 70 55 72 59.5 73.3

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (22.4 mL, 0.333 mol) and a magnetic follower were placedinto a three necked round bottom flask fitted with a pressure equalisingdropping funnel. The system was flushed with nitrogen and placed underpositive pressure. A solution of succinimide terminated poly(MPEG(50)MA)[Mn 4590 PDi 1.22](3.0 g, 9.38×10⁻⁴ mol) dissolved in anhydrousdichloromethane (12 mL) was added to the dropping funnel and thesolution added drop-wise to the ethylenediamine. The solution was leftstirring for 16 hours before dialysing and subsequently freeze drying toisolate the product. ¹H NMR spectra shows the reduction of thesuccinimide O═C—CH ₂—CH ₂—C═O resonance at 2.75 ppm.

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (14.85 mL, 0.222 mol) and a magnetic follower wereplaced into a three necked round bottom flask fitted with a pressureequalising dropping funnel. The system was flushed with nitrogen andplaced under positive pressure. A solution of succinimide terminatedpoly(MPEG(550)MA) [Mn 4590 PDi 1.22](2.0 g, 6.25×10⁻⁴ mol) dissolved inwater (20 mL) was added to the dropping funnel and the solution addeddrop-wise to the ethylenediamine. The solution was left stirring for 16hours before dialysing and subsequently freeze drying to isolate theproduct. ¹H NMR spectra shows reduction of the succinimide O═C—CH₂—CH₂—C═O resonance at 2.75 ppm.

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (20.0 mL, 0.299 mol), water (20 mL) and a magneticfollower were placed into a three necked round bottom flask fitted witha pressure equalising dropping funnel and the solution cooled by placingin an ice bath. The system was flushed with nitrogen and placed underpositive pressure. A solution of succinimide terminatedpoly(MPEG(550)MA) [Mn 4590 PDi 1.22](5.0 g, 1.56×10⁻³ mol) dissolved inwater (50 mL) was added to the dropping funnel and the solution addeddrop-wise to the ethylenediamine. The solution was left stirring for 24hours before dialysing and subsequently freeze drying to isolate theproduct. ¹H NMR spectra shows reduction of the succinimide O═C—CH ₂—CH₂—C═O resonance at 2.75 ppm.

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (1.35 mL, 0.02 mol), water (5 mL) and a magneticfollower were placed into a three necked round bottom flask fitted witha pressure equalising dropping funnel and the solution cooled by placingin an ice bath. The system was flushed with nitrogen and placed underpositive pressure. A solution of succinimide terminatedpoly(MPEG(550)MA) [Mn 4590 PDi 1.22](1.0 g, 3.13×10⁻⁴ mol) dissolved inwater (25 mL) was added to the dropping funnel and the solution addeddrop-wise to the ethylenediamine. The solution was left stirring for 3hours before adding the solution to 2 L of water and dialysing.Subsequently the dialysed solution was freeze dried to isolate theproduct. ¹H NMR spectra shows reduction of the succinimide O═C—CH ₂—CH₂—C═O resonance at 2.75 ppm.

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (1.35 mL, 0.02 mol), anhydrous dichloromethane (5 mL)and a magnetic follower were placed into a three necked round bottomflask fitted with a pressure equalising dropping funnel and the solutioncooled by placing in an ice bath. The system was flushed with nitrogenand placed under positive pressure. A solution of succinimide terminatedpoly(MPEG(550)MA) [Mn 4590 PDi 1.22](1.0 g. 3.13×10⁻⁴ mol) dissolved inanhydrous dichloromethane (25 mL) was added to the dropping funnel andthe solution added drop-wise to the ethylenediamine. The solution wasleft stirring for 3 hours before adding the solution to 2 L of water anddialysing. Subsequently the dialysed solution was freeze dried toisolate the product. ¹H NMR spectra shows reduction of the succinimideO═C—CH ₂—CH ₂—C═O resonance at 2.75 ppm.

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (2.68 mL, 0.04 mol), water (10 mL) and a magneticfollower were placed into a three necked round bottom flask fitted witha pressure equalising dropping funnel and the solution cooled by placingin an ice bath. The system was flushed with nitrogen and placed underpositive pressure. A solution of succinimide terminatedpoly(MPEG(550)MA) [Mn 4590 PDi 1.22](2.0 g, 6.25×10⁻⁴ mol) dissolved inwater (50 mL) was added to the dropping funnel and the solution addeddrop-wise to the ethylenediamine. The solution was left stirring for 5.5hours before adding the solution to 2 L of water and dialysing.Subsequently the dialysed solution was freeze dried to isolate theproduct. ¹H NMR spectra shows reduction of the succinimide O═C—CH ₂—CH₂—C═O resonance at 2.75 ppm.

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (2.67 mL, 0.04 mol), water (10 mL) and a magneticfollower were placed into a three necked round bottom flask fitted witha pressure equalising dropping funnel and the solution cooled by placingin an ice bath. The system was flushed with nitrogen and placed underpositive pressure. A solution of succinimide terminatedpoly(MPEG(550)MA) [Mn 4590 PDi 1.22] (2.0 g, 6.25×10⁻⁴ mol) dissolved inwater (50 mL) was added to the dropping funnel and the solution addeddrop-wise to the ethylenediamine. The solution was stirred for 4 hoursbefore neutralising the solution with 2M HCl and the water subsequentlyremoved using high vacuum. The polymer was dialysed and then freezedried to isolate the product. ¹H NMR spectra shows reduction of thesuccinimide O═C—CH ₂—CH ₂—C═O resonance at 2.75 ppm.

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (0.836 mL, 0.013 mol), water (1 mL) and a magneticfollower were placed into a three necked round bottom flask fitted witha pressure equalising dropping funnel and the solution cooled by placingin an ice bath. The system was flushed with nitrogen and placed underpositive pressure. A solution of succinimide terminatedpoly(MPEG(2000)MA) [Mn 24600 PDi 1.06](5.0 g, 2.03×10⁻⁴ mol) dissolvedin water (100 mL) was added to the dropping funnel and the solutionadded drop-wise to the ethylenediamine. The solution was stirred for 4hours before neutralising the solution with 2M HCl and the watersubsequently removed using high vacuum. The polymer was dialysed andthen freeze dried to isolate the product ¹H NMR spectra shows reductionof the succinimide O═C—CH ₂—CH ₂—C═O resonance at 2.75 ppm.

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (2.5 mL, 0.038 mol), water (10 mL) and a magneticfollower were placed into a three necked round bottom flask fitted witha pressure equalising dropping funnel and the solution cooled by placingin an ice bath. The system was flushed with nitrogen and placed underpositive pressure. A solution of succinimide terminatedpoly(MPEG(2000)MA) [Mn 24600 PDi 1.06](15.0 g, 7.5×10⁻⁴ mol) dissolvedin water (400 mL) was added to the dropping funnel and the solutionadded drop-wise to the ethylenediamine. The solution was stirred for 4hours before neutralising the solution with 2M HCl then adding NaCl (140g) before extracting into dichloromethane (4×150 mL). The organic layerswere combined and dried over Na₂SO₄ filtered and then evaporated todryness before being washed with diethyl ether. The polymer was dialysedand then freeze dried to isolate the product. ¹H NMR spectra showsreduction of the succinimide O═C—CH ₂—CH ₂—C═O resonance at 2.75 ppm.

Conversion of Succinimide End Group of PolyPEG to Amine Group

Ethylenediamine (5.60 mL, 0.08 mol), water (10 mL) and a magneticfollower were placed into a three necked round bottom flask fitted witha pressure equalising dropping funnel and the solution cooled by placingin an ice bath. The system was flushed with nitrogen and placed underpositive pressure. A solution of succinimide terminatedpoly(MPEG(2000)MA) [Mn 21900 PDi 1.21](33.5 g, 1.53×10⁻³ mol) dissolvedin water (500 mL) was added to the dropping funnel and the solutionadded drop-wise to the ethylenediamine. The solution was stirred for 4hours before neutralising the solution with 2M HCl then adding NaCl (140g) before extracting into dichloromethane (4×150 mL). The organic layerswere combined and dried over Na₂SO₄ filtered and then evaporated todryness before being washed with diethyl ether. The polymer was dialysedand then freeze dried to isolate the product. ¹H NMR spectra showsreduction of the succinimide O—C—CH ₂—CH ₂—C═O resonance at 2.75 ppm.

Conversion of Amine End Group of PolyPEG to Maleimide Group

Amine terminated poly(MPEG(550)MA) [Mn 3200](0.5 g, 1.56×10⁻⁴ mol),saturated sodium hydrogen carbonate (2.5 mL) and a magnetic followerwere placed into a three necked round bottom flask and cooled by placingin an ice bath. The system was flushed with nitrogen and placed under aninert atmosphere. To this solution N-methoxycarbonylmaleimide (0.1 g,6.45×10⁻⁴ mol) was added with vigorous stirring. After ten minutes water(5 mL) was added and the reaction left stirring for a further 45minutes. The pH was then adjusted to 3 with 0.5N sulfuric acid and NaCl(0.15 g) was added. The polymer was then extracted in to dichloromethane(3×50 mL), the extracts were combined and dried over Na₂SO4 before beingfiltered and evaporated to dryness. The polymer was then washed withdiethyl ether and dried under vacuum at room temperature. ¹H NMR spectrashows appearance of the maleimide resonances at ˜5.9-6.4 and ˜6.7 ppm.

Conversion of Amine End Group of PolyPEG to Maleimide Group

Amine terminated poly(MPEG(550)MA) [Mn 3200](1.0 g, 3.13×10⁻⁴ mol),saturated sodium hydrogen carbonate (5 mL) and a magnetic follower wereplaced into a three necked round bottom flask and cooled by placing inan ice bath. The system was flushed with nitrogen and placed under aninert atmosphere. To this solution N-methoxycarbonylmaleimide (0.2 g,1.29×10⁻³ mol) was added with vigorous stirring. After ten minutes water(10 mL) was added and the reaction left stirring for a further 45minutes. The pH was then adjusted to 3 with 0.5N sulfuric acid and NaCl(0.30 g) was added. The polymer was then extracted in to dichloromethane(3×50 mL), the extracts were combined and dried over Na₂SO4 before beingfiltered and evaporated to dryness. The polymer was then washed withdiethyl ether and dried under vacuum at room temperature. ¹H NMR spectrashows appearance of the maleimide resonances at ˜5.9-6.4 and ˜6.7 ppm.

Conversion of Amine End Group of PolyPEG to Maleimide Group

Amine terminated poly(MPEG(550)MA) [Mn 3200](1.0 g, 3.13×10⁻⁴ mol),saturated sodium hydrogen carbonate (5 mL) and a magnetic follower wereplaced into a three necked round bottom flask and cooled by placing inan ice bath. The system was flushed with nitrogen and placed under aninert atmosphere. To this solution N-methoxycarbonylmaleimide (0.20 g,1.29×10⁻³ mol) was added with vigorous stirring. After ten minutes water(10 mL) was added and the reaction left stirring for a further 45minutes. The pH was then adjusted to 3 with 0.5N sulfuric acid and NaCl(3.75 g) was added. The polymer was then extracted in to dichloromethane(3×50 mL), the extracts were combined and dried over Na₂SO4 before beingfiltered and evaporated to dryness. The polymer was then washed withdiethyl ether and dried under vacuum at room temperature. ¹H NMR spectrashows appearance of the maleimide resonances at ˜5.9-6.4 and ˜6.7 ppm.

Conversion of Amine End Group of PolyPEG to Maleimide Group

Amine terminated poly(MPEG(2000)MA) [Mn 24600](5.0 g, 2.03×10⁻⁴ mol),saturated sodium hydrogen carbonate (15 mL) and a magnetic follower wereplaced into a three necked round bottom flask and cooled by placing inan ice bath. The system was flushed with nitrogen and placed under aninert atmosphere. To this solution N-methoxycarbonylmaleimide (0.13 g,8.13×10⁻⁴ mol) was added with vigorous stirring. After ten minutes water(15 mL) was added and the reaction left stirring for a further 45minutes. The pH was then adjusted to 3 with 0.5N sulfuric acid and NaCl(7.5 g) was added. The polymer was then extracted in to dichloromethane(4×50 mL), the extracts were combined and dried over Na₂SO4 before beingfiltered and evaporated to dryness. The polymer was then washed withdiethyl ether and dried under vacuum at room temperature. ¹H NMR spectrashows appearance of the maleimide resonances at ˜5.9-6.4 and ˜6.7 ppm.

Conversion of Amine End Group of PolyPEG to Maleimide Group

Amine terminated poly(MPEG(2000)MA) [Mn 24600 PDi 1.06](15.0 g, 6.1×10⁻⁴mol), saturated sodium hydrogen carbonate (45 mL) and a magneticfollower were placed into a three necked round bottom flask and cooledby placing in an ice bath. The system was flushed with nitrogen andplaced under an inert atmosphere. To this solutionN-methoxycarbonylmaleimide (0.38 g, 2.44×10⁻³ mol) was added withvigorous stirring. After ten minutes water (15 mL) was added and thereaction left stirring for a further 45 minutes. The pH was thenadjusted to 3 with 0.5N sulfuric acid and NaCl (7.5 g) was added. Thepolymer was then extracted in to dichloromethane (3×50 mL), the extractswere combined and dried over Na₂SO4 before being filtered and evaporatedto dryness. The polymer was then washed with diethyl ether and driedunder vacuum at room temperature before final purification throughdialysis and isolation by freeze drying. ¹H NMR spectra shows appearanceof the maleimide resonances at ˜5.9-6.4 and ˜6.7 ppm.

Coupling of Succinimide Terminated PolyPEG to Benzylamine

Two different experiments were carried out, each using a differentsolvent. Low molecular weight poly(MPEG(395)MA) (Mn=2700 g/mol,PDI=1.12) (1 g, 0.370×10⁻³ mol) prepared from initiator 7 (i.e.succinimide terminated) and benzylamine (0.40 ml, 3.7×10⁻³ mol) wasdissolved in 10 ml of dry chloroform or distilled water and stirred atroom temperature for 20 hours under nitrogen. After reaction, thesolvent was removed under vacuum by using a rotary evaporator. The crudewas purified by preparative GPC before being precipitated of the polymerin cold Petroleum Ether (40-60° C. Fraction).

Bioconjuction of Succinimide Terminated PolyPEG Polymer

A set of three experiments was carried out, each containing a differentratio polymer/lysozyme. Moreover each set of experiments was left toreact for either 4 hours or 20 hours. Low molecular weightpoly(MPEG(395)MA) prepared from initiator 7 (i.e. succinimideterminated) (Mn=2700 g/mol, PDI=1.12) (41.6 mg, 15.4×10⁻³ mol) for aratio 5/1, (83.2 mg, 30.8×10⁻³ mol) for a ratio 10/1 and (249.5 mg,92.4×10⁻³ mol) for a ratio 30/1 and lysozyme (50 mg, 3.08×10⁻³ mol) wasdissolved in 10 ml of 200 mM phosphate buffer (pH=8) and stirred at 4°C. for 4 hours or 20 hours under nitrogen. The reaction was followed byHPLC in the case of a ratio polymer/lysozyme 30/1. The HPLC system wasfitted with a guard column, a BioSep-SEC-S3000 column and a UV detectorcontinuously measuring the relative absorbance of the mobile phase at215 nm. The system was eluted with 0.1% v/v trifluoroacetic acidsolution in water and acetonitrile (69/31 v/v) at a rate of 0.5 mL/min.In each case, the crude was purified in dialysis bag (Spectra/Porl,MWCO=6-8000 g/mol) and analysed by SDS-PAGE (polyacrylamide resolvinggel cross-linking: 15%, running buffer: 25 mM TRIS base, 250 mM glycine,0.1% SDS, pH 8.7).

Reactions of PolyPEG Polymers Prepared from Initiator 12

Conversion of Acetal End Group of PolyPEG to Aldehyde Group

Acetal-terminated polymer (Mn 11000, PDi 1.15, 3.0 g, 0.27 mmol) wasdissolved in a 1:1 trifluoroacetic acid (TFA)/H₂O solution (100 mL) andthe solution was stirred at room temperature for 48 hours. Most of theacid was removed under reduced pressure and the crude was dissolved inwater and purified by dialysis. The aqueous solution was thenfreeze-dried to give the desired aldehyde terminal polymer (2.8 g, 0.25mmol, 93%) as an off-white solid. (M_(n)˜11,000, PDi 1.13)

Conversion of Acetal End Group of PolyPEG to Aldehyde Group

Acetal-terminated polymer (Mn 22,000, PDi 1.09, 3.0 g, 0.14 mmol) wasdissolved in a 1:1 trifluoroacetic acid (TFA)/H₂O solution (100 mL) andthe solution was stirred at room temperature for 48 hours. Most of theacid was removed under reduced pressure and the crude was dissolved inwater and purified by dialysis. The aqueous solution was thenfreeze-dried to give the desired aldehyde terminal polymer (2.8 g, 1.3mmol, 93%) as an off-white solid. (M_(n)˜22,000, PDi 1.09)

Conversion of Acetal End Group of PolyPEG to Aldehyde Group

Acetal-terminated polymer (Mn=32,000, PDi=1.09, 3.0 g, 0.094 mmol) wasdissolved in a 1:1 trifluoroacetic acid (TFA)/H₂O solution (100 mL) andthe solution was stirred at room temperature for 48 hours. Most of theacid was removed under reduced pressure and the crude was dissolved inwater and purified by dialysis. The aqueous solution was thenfreeze-dried to give the desired aldehyde terminal polymer (2.7 g, 0.084mmol, 90%) as an off-white solid. (M_(n)˜32,000, PDi 1.11)

Bioconjugation of Deprotected PolyPEG Polymers Prepared from Initiator12

Bioconjuction of Aldehyde-Terminated PolyPEG Polymer

Lysozyme (6 mg, 4.2×10⁻⁴ mmol) and aldehyde-terminated polymer(M_(n)˜22,000, PDi 1.09, 110 mg, 0.01 mmol) was dissolved in 5 mL ofacetate/acetic acid buffer (pH=5) and 0.15 mL of NaCNBH₃ (0.25 mM inwater) was added dropwise. The solution was stirred at room temperatureand samples were taken at regular intervals. The reaction was monitoredby HPLC fitted with a guard column, a bioSep-SEC-S3000 column and an UVdetector.

Bioconjuction of Aldehyde-Terminated PolyPEG Polymer

Lysozyme (6 mg, 4.2×10⁻⁴ mmol) and aldehyde terminated polymer(M_(n)˜22,000, PDi 1.09, 110 mg, 0.01 mmol) was dissolved in 5 mL ofphosphate buffer (pH=6) and 0.15 mL of NaCNBH₃ (0.25 mM solution inwater) was added dropwise. The solution was stirred at room temperatureand samples were taken at regular intervals. The reaction was monitoredby HPLC fitted with a guard column, a bioSep-SEC-S3000 column and an UVdetector.

Reactions of PolyPEG Polymers Prepared from Initiator 14

Conversion of BOC End Group of PolyPEG to Amine Group

BOC terminated polymer (M_(n)=6400 g mol⁻¹, 3.2 g, 1.0 mmol) wasdissolved in CH₂Cl₂ (25 mL), trifluoroacetic acid (3.9 mL, 50 mmol) wasadded via syringe and the resulting solution was stirred at roomtemperature for 16 h. The solvent was then removed under reducedpressure and the resulting orange-brown oil was dissolved in deionizedwater and dialyzed. The polymer solution was freeze-dried. Toluene (50mL) was then added and the solvent was removed under reduced pressure.This procedure was repeated three times and the expected amineterminated polymer as the trifluoroacetic acid salt (2.5 g, 0.81 mmol,81% yield) was obtained as a yellow-orange oil. ¹H NMR revealed thecomplete disappearance of the singlet relative to the Boc group at 1.4ppm. M_(w)/M_(n) (GPC)=1.11

Conversion of Amine End Group of PolyPEG to Malemide Group

3-Maleimidepropionyl chloride (13.0 mmol) was dissolved in 100 mL ofCH₂Cl₂, diisopropylethylamine (DIPEA, 2.3 ml, 13.0 mmol) was added viasyringe and the solution was cooled to 0° C. A solution of amineterminated polymer as the trifluoracetic acid salt (1.5 g, 0.47 mmol) in30 mL of CH₂Cl₂ was added dropwise (ca. 15 min) and the mixture wasstirred at 0° C. for 1 h, then at room temperature for 2 days. Thesolvent was then removed under reduced pressure and 200 mL of water wereadded to the brown residue. The suspension was centrifugate and purifiedby dialysis (Millipore, regenerated cellulose, MWCO 1 kDa, filtrationarea 0.23 m²).

The polymer solution was freeze-dried. Toluene (50 mL) was then addedand the solvent was removed under reduced pressure. This procedure wasrepeated three times and the expected malemide terminated polymer wasobtained as a pale yellow oil. A conversion of 80% can be calculated by¹H NMR, comparing the integration of the vinylic protons of themaleimide moiety and that of the terminal OCH₃ of the PEG side-chains.Mw/Mn (GPC)=1.06.

Reactions of PolyPEG Polymers Prepared from Initiator 15Conversion of Furan End Group of PolyPEG to malemide Group

A solution of polymer prepared from initiator 15 (3.0 g, 0.36 mmol) intoluene (25 mL) was warmed to reflux and the reaction was monitored by¹H NMR analysis on samples taken at regular intervals of time. After 7 hthe solvent was removed under reduced pressure to give the maleimideterminated polymer as a pale orange oil. Comparison of the integrationof the maleimide vinyl signals and the terminal OCH₃ of the PEGside-chains confirmed that the maleimide function did not decomposeduring the deprotection step.

REFERENCES

-   1. D. M. Haddleton, M. C. Crossman, B. H. Dane, D. J. Duncalf, A. M.    Henning, D. Kukulj and A. J. Shooter, Macromolecules, 1999, 32,    2110.-   2. R. N. Keller and W. D. Wycoff, Inorg. Synth., 1947, 2, 1.-   3. James R. Dudley, Jack T. Thurston, Frederic C. Schaefer, Dagfrid    Holm-Hansen, Clarence J. Hull, and Pierrepont Adams, J. Am. Chem.    Soc., 1951, 73, 2986.

1. (canceled)
 2. A comb polymer having the general formula:A-(D)_(d)-(E)_(e)-(F)_(f) where: A is a moiety capable of covalentlybinding to a protein, a polypeptide, a fat or a carbohydrate, providedthat A is not a hydroxyl group; (D)_(d) is a polymeric unit produced bypolymerizing a plurality of monomer molecules selected from one or moreolefinically unsaturated monomers by addition polymerization, whereinsaid monomers are not the monomers used to produce (E)_(e); (E)_(e) is apolymeric unit comprising an alkoxy polyether, said polymeric unitproduced by polymerizing by addition polymerization a plurality ofmonomer molecules which are linear, branched, or star-shaped,substituted or non-substituted and have an olefinically unsaturatedmoiety; (F)_(f), is a polymeric unit produced by polymerizing byaddition polymerization a plurality of monomer molecules selected fromone or more olefinically unsaturated monomers which are not as definedin E; d and f are each independently an integer between 0 and 500; and eis an integer of 10 to
 1000. 3. A comb polymer according to claim 2,wherein (E)_(e) comprises a poly(alkylene) glycol orpolytetrahydrofuran.
 4. A comb polymer according to claim 2, having anaverage molecular weight of 2,000 to 80,000.
 5. The comb polymeraccording to claim 2, wherein the monomers which are polymerized toproduce each of (D)_(d), (E)_(e), and (F)_(f) comprise an olefinicallyunsaturated group independently selected from acrylate, methacrylate,methylmethacrylate, styrene, methylacrylate and diene.
 6. The combpolymer according to claim 2, wherein the poly (alkylene glycol) ispoly(ethylene glycol) (PEG) or poly(propylene glycol).
 7. The combpolymer according to claim 2, wherein the moiety capable of binding tothe protein, the polypeptide, the nucleic acid, the carbohydrate or thefat comprises succinimidyl succinate, N-hydroxysuccinimide, succinimidylpropionate, succinimidyl butanoate, triazine, vinyl sulfone,propionaldehyde, acetaldehyde, tresylate, benzotriazole carbonate,maleimide, pyridyl sulfide, iodoacetamide or succinimidyl carbonate. 8.A comb polymer according to claim 2, wherein the moiety which is capableof reacting with the protein or the polypeptide comprises:

where n=integer of 0 to 10;

where m=integer of 0 to 10, Y is an aliphatic or aromatic moiety; and

where R′ is H, methyl, ethyl, propyl or butyl, and X=halide.
 9. The combpolymer according to claim 2 comprising a fluorescent label.
 10. Thecomb polymer according to claim 9, wherein the fluorescent label is acoumarin.
 11. A polymer-biomolecule conjugate produced by a methodcomprising the step of reacting a protein or a polypeptide with the combpolymer of claim 2, thereby forming the polymer-biomolecule conjugate.12. The polymer-biomolecule of claim 11, which is biologically-active.13. A compound according to claim 11, which is a drug.
 14. Apharmaceutical composition comprising the polymer-biomolecule conjugateof claim 11 and a pharmaceutically acceptable carrier.
 15. A combpolymer produced by a method comprising the steps of: (a) providing: (i)a plurality of monomers which are linear, branched or star-shaped,substituted or non-substituted, and have an olefinically unsaturatedmoiety, the olefinically unsaturated moiety being capable of undergoingaddition polymerization, wherein the monomers comprise alkoxypolyethers; (ii) an initiator compound; the initiator compoundcomprising a homolytically cleavable bond, wherein said initiatorcompound comprises a moiety which when attached to the comb polymer iscapable of binding to a protein, polypeptide, carbohydrate, fat ornucleic acid, wherein said moiety is optionally protected, provided thatthe initiator compound is not 2-hydroxyethyl-2′-bromopropionate; and(iii) a catalyst capable of catalyzing the polymerisation of themonomer, and (b) causing the catalyst to catalyse, in combination withthe initiator, the polymerisation of a plurality of the monomers toproduce the comb polymer, and (c) optionally deprotecting the moietywhich when attached to the comb polymer is capable of binding to aprotein, polypeptide, carbohydrate, fat or nucleic acid.
 16. The combpolymer of claim 15, wherein the alkoxy polyether is poly(alkyleneglycol) or polytetrahydrofuran.
 17. The comb polymer of claim 15,wherein the olefinically unsaturated moiety is acrylate, methacrylate,methylmethacrylate, styrene, methylacrylate, or a diene.
 18. The combpolymer of claim 15, wherein the initiator compound is selected from:A-S—C(O)—R, A-S—C(S)—O—R, R—S—C(O)-A, R—S—C(S)—O-A, A-B—X,

wherein X is a halide; R is C₁ to C₂₀ substituted or non-substituted,straight chain, branched chain, cyclic, heterocyclic or aromatic alkyl;A is an optionally protected moiety which, when attached to the combpolymer, is capable of binding to a protein or polypeptide; and B is alinker or a direct bond.
 19. The comb polymer of claim 18, wherein A isselected from succinimidyl succinate, N-hydroxysuccinimide, succinimidylpropionate, succinimidyl butanoate, triazine, vinyl sulfone,propionaldehyde, acetaldehyde, tresylate, benzotriazole carbonate,maleimide, pyridyl sulfide, iodoacetamide and succinimidyl carbonate.20. The comb polymer of claim 15, wherein the initiator compound isselected from the group consisting of:

where n is an integer of 0 to 10, and X is a halide.